Nanodiamond particles and related devices and methods

ABSTRACT

Nanodiamond particles and related devices and methods, such as nanodiamond particles for the detection and/or quantification of analytes, are generally described. In some embodiments, the device comprises a plurality of nanodiamond particles and a species bound to the nanodiamond particles. In certain embodiments, the plurality of nanodiamond particles may be exposed to a sample suspected of containing an analyte. In some cases, the analyte may bind to the species such that the presence of the analyte in the sample may be detected. In some embodiments, the devices, systems, and methods described herein are useful for the detection of an analyte in a sample obtained from a subject for, for example, diagnostic purposes. In some cases, the systems, devices, and methods described herein may be useful for diagnosing, prevent, treating, and/or managing a disease or bodily condition. In an exemplary embodiment, such systems, devices, and methods described herein may be useful for detecting and/or quantifying the presence of a virus (e.g., ebola) in a subject and/or a sample obtained from the subject.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 62/383,657, filed Sep. 6, 2016, and entitled“Engineering And Utility Of Fluorescent Nanodiamond Particles (F-NDP)For Diagnostics And Treatment Of Blood Clots In Human And VeterinaryMedicine,” and to U.S. Provisional Application No. 62/522,036, filedJun. 19, 2017, and entitled “Nanodiamond Particles And Related DevicesAnd Methods,” each of which is incorporated herein by reference in itsentirety for all purposes.

TECHNICAL FIELD

Nanodiamond particles and related devices and methods, such asnanodiamond particles for the detection and/or quantification ofanalytes, are generally described. In addition, the present inventionrelates to the field of medical and veterinary diagnostics andtreatment. More specifically, the invention relates to a diagnosticreagent, tool, and system that are specific for detection of plateletsand blood clots. In addition, the invention relates to detection ofinternal body bleeding sites in a variety of diseases and traumaconditions.

BACKGROUND

Cardiovascular diseases, such as stroke and heart attack, are theleading cause of mortality in developed countries. Deaths from strokesand heart attacks are predominantly the consequence of blood clots(thrombi) formed in the cerebral and cardiac vessels or thrombo-embolicevents (TEE) associated with blood clots formed in remote vessels (e.g.,peripheral venous system, cardiac atria appendixes). While severalfactors are well known to contribute to a fatal TEE (e.g.,atherosclerosis vascular disease) there is a clear “diagnostic andprognostic gap” in the assessment of the specific and “total clotburden” in individuals that carry known risk factors (e.g.,atherosclerotic vascular disease), let alone factors not yet fullyvetted as predictive of TEE. Invariably, clinical presentation ofconsequences of blood flow occlusion by thrombi leading to stroke orheart attack command prompt medical investigations in search for the TEEculprits. Such investigations are mostly hospital-based imaging tests,such as angiography, CAT scans, MRI, and ultrasound. The currenttechnologies are important for timely and successful management ofstrokes and heart attacks, yet several important limiting factors mustbe addressed. First, the general population, especially elderly peoplewho carry cardiovascular risks, often have limited access tohospital-based technologies to assess blood clots in vessels. Second,even in hospitals, access to these imaging technologies has a certaintime requirement associated with the tests and their evaluation byspecialists. In the case of a stroke, where the treatment window islimited to three to four and a half hours after the onset of the event,much of the time is spent in establishing patient eligibility forthrombolysis treatment, often to the extent of missing the criticalwindow for treatment.

On the other extreme, once a diagnosis of cardiac arrhythmia, such asatrial fibrillation (chronic, relapsing) is made, the risk of TEEmandates lifelong treatment with anti-coagulants even though thepresence of clots in the cardiac chambers (appendixes) is unknown. Thesefew examples point to a major “diagnostic gap” of TEE risks, which dueto lack of early diagnosis and preventative measures often results infatal outcomes. Early assessment of whole body clot burden or TEE risk,in ambulatory settings, that allows easy and broad access and affordablecost, is needed.

Furthermore, immunochromographic assays, such as lateral flow assays,are generally used to detect the presence or absence of an analyte suchas an antigen in a sample. However, such assays generally lack automatedprocessing, accurate quantification methods, and may, in some cases,require subjective interpretation, leading to false positives and/orfalse negatives.

Accordingly, improved devices and methods are needed.

SUMMARY

Nanodiamond particles and related devices and methods, such asnanodiamond particles (e.g., fluorescent nanodiamond particles) for thedetection and/or quantification of analytes, are generally described.The subject matter of the present invention involves, in some cases,interrelated products, alternative solutions to a particular problem,and/or a plurality of different uses of one or more systems and/orarticles.

In one aspect, diagnostic agents are provided. In some embodiments, thediagnostic agents comprise a fluorescent nanodiamond particle chemicallybonded to a polypeptide and/or polynucleotide.

In another aspect, fluidic devices are provided. In some embodiments,the fluidic device comprises a sample inlet, a reservoir in fluidiccommunication with the sample inlet, the reservoir comprising aplurality of fluorescent nanodiamond particles, a plurality of a firstspecies bound to the plurality of fluorescent nanodiamond particles, adetection region in fluidic communication with the reservoir, thedetection region comprising a plurality of a second species bound to thedetection region, and a control region in fluidic communication with thedetection region, the control region comprising a plurality of a thirdspecies bound to the control region.

In yet another aspect, systems are provided. In some embodiments, thesystem comprises a sample inlet, a reservoir in fluidic communicationwith the sample inlet, the reservoir comprising a plurality offluorescent nanodiamond particles, a plurality of a first species boundto the plurality of fluorescent nanodiamond particles, a detectionregion in fluidic communication with the reservoir, the detection regioncomprising a plurality of a second species bound to the detection regionand a detector configured to quantify a fluorescent emission at thedetection region.

In some embodiments, the system comprises a sample inlet, a reservoir influidic communication with the sample inlet, the reservoir comprising aplurality of fluorescent nanodiamond particles, a plurality of a firstspecies bound to the plurality of fluorescent nanodiamond particles, adetection region in fluidic communication with the reservoir, thedetection region comprising a plurality of a second species bound to thedetection region and a detector configured to quantify an infraredsignal at the detection region.

In yet another aspect, methods are provided. In some embodiments, themethod comprises introducing, into a fluidic channel of a fluidicdevice, a sample suspected of containing an analyte, exposing the sampleto a species bound to a plurality of fluorescent nanodiamond particlessuch that the analyte, if present, binds to at least a portion of thespecies bound to the plurality of fluorescent nanodiamond particles,removing any fluorescent nanodiamond particles and species not bound tothe analyte, and quantifying a fluorescence emission of the plurality offluorescent nanodiamond particles bound to the analyte.

In some embodiments, the method comprises administering, to a subject(e.g., a human, a mammal) suspected of having a particular analyte, aplurality of fluorescent nanodiamond particles bound to a species suchthat the species may bind to the analyte, if present, and detecting afluorescent emission of the plurality of fluorescent nanodiamondparticles comprising the species bound to, if present, the analyte.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIG. 1A is a schematic illustration of a system including fluorescentnanodiamond particles, according to one set of embodiments;

FIG. 1B is a schematic illustration of a system including fluorescentnanodiamond particles, according to one set of embodiments.

FIG. 2A is a schematic illustration of a system including fluorescentnanodiamond particles for detection of an analyte, according to one setof embodiments.

FIG. 2B is a schematic illustration of a system including fluorescentnanodiamond particles for detection of an analyte upon introduction of asample suspected of containing the analyte, according to one set ofembodiments.

FIG. 2C is a schematic illustration of a system including fluorescentnanodiamond particles for detection of an analyte after introduction ofa sample suspected of containing the analyte, according to one set ofembodiments.

FIG. 3 is a schematic illustration of an exemplary fluorescentnanodiamond particle functionalized with a species such as human IgG,according to one set of embodiments.

FIG. 4 is a photograph of an exemplary system including a reservoircomprising a plurality of fluorescent nanodiamond particles, accordingto one set of embodiments.

FIG. 5 is a plot of semi-ELISA for detection of human IgG bound tofluorescent nanodiamond particles at various concentrations versuscontrol (bovine serum albumin (BSA)), according to one set ofembodiments.

FIG. 6 is a plot of the quantification of fluorescent nanodiamondparticles functionalized with bitistatin in a carotid artery clot versuscontrol vessels in rats, according to one set of embodiments.

FIG. 7A shows scanning confocal microscopy images of carotid arteriesfrom rats with generated clots with fluorescent nanodiamond particlesintroduced, according to one set of embodiments.

FIG. 7B shows a scanning confocal microscopy image of a carotid arteryfrom a rat with a generated clot with fluorescent nanodiamond particlesintroduced, according to one set of embodiments.

FIG. 8 shows images of tissue suspensions of clots treated or not withfluorescent nanodiamond particles, injected systematically to thefemoral vein close to a clot generated in carotid artery, according toone set of embodiments;

FIG. 9 is a fluorescence scan result showing two dimensional screeningof excitation vs. emission wavelengths to optimize fluorescence ofNDP-Fs used in the exemplary embodiment. The figure shows optimizationof the maximal fluorescence for NDP-Fs. The heat map represents theresults of fluorescence screening in the entire spectrum of excitationvs. emission wavelengths. The experiment was performed using a Tecanplate reader. NDP-Fs were applied on a 96-well plate (0.3 mg/0.1 ml) asa suspension in PBS. The blank, or negative control, was establishedusing PBS alone.

FIG. 10 presents a line graph indicating detection of Bt on NDP-Fs in a“semi-ELISA” assay. NDPs were coupled to Bt or BSA, which were used inconcentrations as indicated on the X-axis, per 1 mg of NDP-Fs. 0.2 mg ofeach NDP sample was used for semi-ELISA in three replicates. Theexperiment was performed on a 96-well plate (U-shape bottom) with gentlerotation during incubations. NDPs were blocked with 10% goat serumbefore primary antibody against Bt was added. NDP samples were incubatedwith anti-Bt antibody for one hour at 37° C., and the plate was washedthree times with PBST by centrifugation (1,000×g) at room temperature.Goat anti-rabbit IgG AP conjugated (Sigma Inc.), diluted 1:2000, wasadded and incubated for one hour as above. Final washing was performedas above and substrate (pNPP) was added to AP. Color was developed for30 minutes, and NDP samples were centrifuged. The supernatant wastransferred to a 96-well plate and read using an ELISA plate readerunder 405 nm wavelength. Error bars represent standard deviation (SD)from three independent samples applied for the semi-ELISA procedure.

FIG. 11 is a line graph showing interaction of purified integrin withNDPs in semi-ELISA. The experiment was performed using a U-shape bottom96-well plate. The plate was blocked overnight with 5% BSA, whereas NDPswere blocked with 3% BSA by incubation for 1 hour at 37° C. beforeapplication on the plate. Blocked NDPs were added to the wells (0.2 mgper well) and the plate was centrifuged (10,000×g) at room temperature.Platelet Fibrinogen Receptor (PFR) at the indicated concentrations wasadded in 0.1 ml of Hanks' Balanced Salt Solution (HBSS) containing Ca²⁺(as CaCl₂)) and Mg²⁺ (as MgCl₂) at physiological concentrations to eachwell and incubated for 1 hour at 37° C. NDPs were washed three times bycentrifugation of the plate (1,000×g) at room temperature and primarypolyclonal antibody against the fibrinogen receptor was added (2 μg/ml).Incubation was performed for one hour at 37° C., and samples were washedthree times, as described above. Goat anti-rabbit IgG AP conjugated(Sigma Inc.), diluted 1:3000, was added and incubated for one hour asabove. Final washing was performed as above and substrate (pNPP) wasadded to AP. Color was developed for 30 minutes, and NDP samples on theplate were centrifuged at 10,000×g at room temperature to collect theNDP as a pellet. Supernatant was transferred to flat bottom 96-well (0.1ml) plates and read using an ELISA plate reader at 405 nm wavelength.

FIG. 12 presents a line graph showing adhesion of NDP-F-Bt and NDP-BSAto immobilized PFR. PFR was immobilized on a 96-well plate by overnightincubation at 4° C. in PBS. The plate and NDPs were blocked with 3% BSA.NDPs coupled to 1 mg protein (Bt or BSA) were used in the experiment.NDPs (300 mg) were added to the wells. Incubation was performed for onehour at 37° C. in HBSS buffer containing calcium and magnesium atphysiological concentrations. Unbound NDPs were intensively washed outsix times using the same buffer with vacuum aspiration. Finally, HBSS(100 μl) was added to the wells and fluorescence was read using afluorescence plate reader with 485 nm (excitation) and 530 (emission)wavelengths.

FIG. 13 presents a line graph showing quantitation of adhesion ofNDP-F-Bt and NDP-BSA to immobilized fibrinogen receptor. Fibrinogenreceptor was immobilized on 8-well glass chamber slides by overnightincubation at 4° C. The wells were blocked with 3% BSA, and NDPspreviously also blocked by 3% BSA were added (50 mg per well per 200 ml)in HBSS containing calcium and magnesium at physiologicalconcentrations. The adhesion procedure was performed as per FIG. 12. Inthe final step, the slide was prepared with mounting buffer (VectorLab). Images were analyzed under fluorescence microscope (400×) using anoil objective. The numbers of NDPs were calculated using ImageJsoftware. Error bars represent SD for three independent pictures takenfor each concentration of fibrinogen receptor.

FIG. 14 depicts representative images of adhered NDP-F-Bt to immobilizedPFR. PFR (concentrations indicated) was immobilized on an 8-well chamberslide, and the experiment was performed as described in FIG. 13. In thelegend, the bars represent 20 μm.

FIG. 15 shows pictures of fluorescent images taken by IVIS and confocalmicroscopy of carotid artery clots after treatment with FNDP via anexternal carotid artery infusion. FIG. 15A shows an in situ carotidbifurcation region image indicating fluorescence of a carotid arterialclot after treatment visible via IVIS imaging after exposure of thecarotid bifurcation zone. FIGS. 15B and 15C are high magnificationimages of fluorescence emanating from the carotid bifurcation in vivosuggesting accumulation of FNDP in the clot. FIG. 15D shows ex vivofluorescence of carotid artery bifurcation denoting one branch thatshows fluorescence corresponding to the clot location within the carotidbifurcation. FIGS. 15E and 15F show confocal images taken on an OlympusIX83, showing that FNDP fluorescence is detected at an excitation of 543nm and an emission of 655-755 nm.

FIG. 16 shows fluorescent images taken by IVIS and confocal microscopyof carotid artery clots after intravenous treatment with FNDP. FIG. 16Ashows an ex vivo fluorescent image of a carotid artery fromsaline-treated control. FIG. 16B shows an ex vivo fluorescent image of acarotid artery from an IV FNDP-treated animal showing fluorescencelocalized to the branch with clot. FIGS. 16C-F show confocal imagestaken on an Olympus IX83. FIG. 16G is a graph showing the number ofFNDPs present in carotid clot lysates from animals treated locally viathe external carotid artery or intravenously as compared with salinetreated controls.

FIGS. 17A-17B show fluorescent microscopy of the specificity of theinteraction of F-NDP-Bt for clot generation from rat blood plasma bythrombin (1 U/ml). FIG. 17A shows images of plasma clots obtained fromfluorescence microscope Olympus IX81 analysis, under 100× magnification.FIG. 17B shows images of plasma clots obtained in an IVIS 50 imagingsystem. Wavelengths used for measurement: excitation Cy5.5 BkG (580-610nm), emission Cy5.5 (695-770 nm). For background subtraction: excitationGFP (445-490 nm), emission Cy5.5 (695-770 nm). Exposure time: 1 minute.Arrows point the localization of the clot.

FIGS. 18A-18B show images of vessels filled with F-NDP-Bt implantedsubcutaneously in a rat (post-mortem). FIG. 18A shows an image of theimplanted glass capillaries filled with F-NDP-Bt (4 mg/ml) or PBS(control). Exposure time was 5 seconds. FIG. 18B shows an image of a rataorta filled with F-NDP-Bt. The rat aorta was dissected from aeuthanized female rat, washed with PBS to remove residues of coagulatedblood, and filled with 300 μl of F-NDP-Bt suspension (2 mg/ml) in PBS.

FIG. 18C shows plots of average fluorescence intensity for F-NDP-Bt,F-NDP-Bt+lotrafiban (lt), and F-NDP-BSA in thrombin-induced PRP clots.

FIG. 18D shows representative images of the clots in FIG. 18C from IVIS.

FIGS. 19A-19C show dose response curves with and without 700 nm diameterfluorescent nanodiamond particles functionalized with bitistatin (i.e.probe) for mean maximum platelet aggregation+/−standard deviation linearregression lines for proteinase-activated receptor 4 (PAR4 AP, FIG.19A), adenosine diphosphate (ADP, FIG. 19B), and arachidonic acid (AA,FIG. 19C).

FIGS. 20A-20C show box plots of 700 nm and 200 nm diameter probesbinding to platelet populations at various concentrations. FIG. 20Ashows the percentage of all platelets, FIG. 20B shows the percentage ofCD62 +ve platelets, and FIG. 20C shows the percentage of GPIIb/IIIa +veplatelets bound to the 700 nm and 200 nm probes at variousconcentrations. Data shown are the mean (+), the line within the boxrepresents the median, upper and lower edges of the box represents75^(th) and 25^(th) percentiles, and upper and lower whiskers representthe 95^(th) and 5^(th) percentiles.

FIG. 21 shows a plot of percentage of platelets bound to 700 nm diameterprobes for stimulated versus unstimulated platelet populations. Datashown are mean percentage (+/−standard deviation) of all platelets boundto the probe in simulated versus unsimulated platelets at aconcentration of 350 mcg/mL of probe.

FIG. 22 shows a plot of percentage of platelets bound to 200 nm diameterprobes for stimulated versus unstimulated platelet populations. Datashown are mean percentage (+/−standard deviation) of all platelets boundto the probe in simulated versus unsimulated platelets at aconcentration of 350 mcg/mL of probe.

FIGS. 23A-23D show flow cytometry dotplots for unsimulated and simulatedpopulations of cells and platelets.

FIGS. 24A-24D show flow cytometry dotplots for unsimulated and simulatedpopulations of cells and platelets.

FIGS. 25A-25B show plots of volume versus size (FIG. 25A) andfluorescence intensity versus size (FIG. 25B) for F-NDPs before andafter sterilization.

FIGS. 26A-26C show a comparison of NIR fluorescence intensity ofF-NDP(NV) and F-NDP(NVN) in suspensions. (FIG. 26A) F-NDP suspensionswere scanned for fluorescence in a fluorescence plate reader for a rangeof excitation and emissions. The fluorescence is normalized bysubtracting a blank well and log 10 processed. (FIG. 26B) Capillarieswere filled with indicated density of F-NDP in PBS and analyzed by IVISimaging using 5 seconds exposure. Insert indicates representative imagesof capillaries. Average fluorescence is presented in the plot. (FIG.26C) Comparison of fluorescence intensity for different concentrationsof F-NDP(NV) as function of exposure time. Error bars represent SD forthree to five independent experiments. *Difference between F-NDP(NV) andF-NDP(NVN) (P<0.01)

FIGS. 27A-27D show a comparison of intensity of fluorescence ofdifferent sizes of F-NDP(NV) under different exposure times in IVIS.Left panes shows representative images of F-NDP(NV), presented inconcentrations as pointed on the plot. Sizes of particles are (from thetop): 100, 700, and 10,000 nm, respectively. Error bars represent SD forthree independent experiments.

FIGS. 28A-28H show a comparison of the ability to detect F-NDP NIRfluorescence through different biological barriers using IVIS. (FIG.28A) Capillaries filled with F-NDP(NV) (4 mg/ml), F-NDP(NVN) (4 mg/ml),or PBS were positioned under abdominal skin patch of euthanized rat.Positions of capillaries are indicated by arrows. (FIG. 28B) Capillaryfilled with F-NDP(NV) (4 mg/ml) covered by rat quadriceps muscles rangedfrom 2 mm (flanking) to 5.9 mm (in the center). (FIG. 28C) Capillariesfilled with F-NDP (4 mg/ml) or PBS were inserted into porcine axillaryvein. (FIG. 28D) Capillaries filled with F-NDP (4 mg/ml) or PBS werecovered with porcine skin (2.5 mm) free of subdermis (FIG. 28E)Intensity of fluorescence for different concentrations of F-NDP(NV)through 2.5 mm porcine skin free of subdermis. (FIG. 28F) Porcineaxillary veins filled with F-NDP(NV) (2 mg/ml) or PBS and covered with 8mm porcine skin including dermis and subdermis. (FIG. 28G) Capillariesfilled with F-NDP(NV) (20 mg/ml), F-NDP(NVN) (20 mg/ml) and PBS (samevolume) covered with porcine skin containing increased thickness ofadipose tissue (presented on the insert). (FIG. 28H) Representativeultrasound of human carotid artery showing the artery 11.89 mm below thesurface.

FIGS. 29A-29N show F-NDP(NV)-Bit infusion via external carotid artery.Carotid arteries clots are imaged in situ and removed from the animalfor further direct imaging and analysis. (FIGS. 29A-29F) Images offluorescence recorded by an IVIS scanner designed for whole animalimaging using a 580-610 nm excitation and a 695-770 nm emission passbandwith 2 second exposure. Auto-fluorescence was subtracted based onexcitation at 445-490 nm (FIG. 29A, FIG. 29B) In situ fluorescenceimaging of carotid arterial clot after treatment in duplicate animals byIVIS (separation of neck particle by dissection) Scale bar=1 cm. (FIGS.29C-29F) Ex-vivo fluorescence of isolated carotid artery afterF-NDP(NV)-Bit treatment (FIG. 29C, FIG. 29D) or with vehicle control ofsaline (FIG. 29E, FIG. 29F). Scale bar=1 mm. (FIGS. 29G-29J) Confocalimage stacks were taken on Olympus FV 1000. F-NDP(NV)-Bit fluorescencewas detected at an excitation of 543 nm and an emission of 655-755 nm.Background fluorescence was collected from the same excitation, withemissions of 555-625 nm and was subtracted from the foreground. Scalebar=1 mm. Fluorescence of carotid artery after F-NDP(NV)-Bit treatment(FIG. 29G, FIG. 29H) or with saline treatment (FIG. 29I, FIG. 29J).(FIG. 29K) In situ images of a FeCl3-generated clot carotid arterycompared with untreated contralateral artery. (FIGS. 29L-29N) Clotsdissolved with RIPA lysis buffer and replicates are combined together toform a lysate. Aliquots of the lysate was then deposited onto a coverglass (10 μL) and imaged with 20× objective. Scale bar=100 □m. (FIG.29K) Large numbers of F-NDP(NV)-Bit are visible. (FIG. 29L) After IVtreatment at low dose (1 mg), F-NDP(NV)-Bit are found in the lysate atthe site of clot formation. (FIG. 29N) Almost no fluorescent particlesare detected in saline control.

FIGS. 30A-30S shows F-NDP(NV)-Bit intravenous infusion. (FIGS. 30A-30J)images of fluorescence are performed on an IVIS scanner designed wholeanimal imaging using a 580-610 nm excitation and a 695-770 nm emissionpassband with 2 second exposure. Autofluorsesence was subtracted basedon excitation at 445-490 nm. (FIGS. 30A-30C) Gross image indicatingfluorescence of carotid arterial clot after treatment in triplicateanimals is visible via IVIS imaging after exposure of artery. Scalebar=1 cm. (FIGS. 30D-30I) Ex-vivo fluorescence of carotid artery afterF-NDP(NV)-Bit treatment (FIGS. 30D-30F) or with saline treatment (FIGS.30G-30I) Scale bar=1 mm. (FIGS. 30J-30O) Confocal image stacks taken onOlympus FV 1000. F-NDP(NV)-Bit fluorescence is detected at an excitationof 543 nm and an emission of 655-755 nm. Scale bar=1 mm. Fluorescence ofcarotid artery after F-NDP(NV)-Bit treatment (FIGS. 30J-30L) or withsaline treatment (FIGS. 30M-30O). (FIG. 30P) Lysates from solubilizedcarotid arteries are imaged and F-NDP(NV)-Bit are counted byhemocytometer. Contralateral untreated control is presented in insert.Scale bar=100 m (FIG. 30Q) Total number of F-NDP(NV)-Bit detected percarotid bifurcation in the clotted side compared with the contralateralcontrol as counted by hemocytometer. (FIG. 30R, FIG. 30S) Brightnessafter subtraction of background via IVIS (FIGS. 30D-30I) and LSCM (FIGS.30J-30O) imaging of treated and contralateral untreated carotidbifurcations. Error bars represent standard deviation. *=p<0.05,**=p<0.01 vs control by t-test.

DETAILED DESCRIPTION

Nanodiamond particles and related devices and methods, such asnanodiamond particles for the detection and/or quantification ofanalytes, are generally described. In some embodiments, the presentinvention provides diagnostic agents and methods for risk assessment ofsubjects, including both humans and non-human animals at risk ofthrombo-embolic events (TEE), which are the main cause of cardiovasculardeath from strokes and heart attacks. The diagnostic agents and methodsof the invention enable assessment of the total body burden ofintravascular clots and detection of nascent thrombi at commonpredilection sites for clot formation using non-radiation (e.g., X-ray),non-MRI, and non-ultrasound techniques. The technology generallycomprises a non-invasive, telemetry-based fluorescent recording systemsuitable for use in a fast and affordable ambulatory setting. Theinvention includes multiple innovative scientific and engineeringbreakthroughs.

The invention is based, at least in part, on the recognition thatfluorescent nano-diamond particles (NDP-F) functionalized with a species(e.g., a polypeptide such as the disintegrin Bitistatin (Bt),immunoglobulins such as IgG, and/or bovine serum albumin (BSA)) may havethe innate ability to bind e.g., avidly to activated platelet fibrinogenreceptors (PFR). For example, data presented herein demonstratesuccessful coupling of Bt to NDP-F and retention of Bt bioactivity. Themethods, reagents, and de novo protocols used to accomplish theinvention are not known to the inventors as having been reported byothers before.

In some embodiments, the device comprises a plurality of nanodiamondparticles and a species (e.g., a polypeptide, a polynucleotide) bound tothe nanodiamond particles. In certain embodiments, the plurality ofnanodiamond particles may be exposed to a sample suspected of containingan analyte. In some cases, the analyte may bind to the species such thatthe presence of the analyte in the sample may be detected. In someembodiments, the devices, systems, and methods described herein areuseful for the detection of an analyte in a sample obtained from asubject for, for example, diagnostic purposes. In some cases, thesystems, devices, and methods described herein may be useful fordiagnosing, prevent, treating, and/or managing a disease or bodilycondition. In an exemplary embodiment, such systems, devices, andmethods described herein may be useful for detecting and/or quantifyingthe presence of a virus (e.g., ebola) in a subject and/or a sampleobtained from the subject.

Advantageously, as compared to traditional systems for quantifyinganalytes (e.g., viruses, bacteria, toxins, environmental pollutants,etc.) in a sample from a subject, the systems and methods describedherein may comprise quantification of the amount of analyte present inthe sample.

The present invention may also enable broad scale survey of TEE risksthat may be applicable in many medical emergency and life-threateningconditions beyond strokes and heart attacks. The technology not only canbe used for individuals suspected to be at risk for TEE but also can beperiodically deployed as part of primary health care office assessments,no different than annual mammography, lipids tests, or physicalexaminations. For example, in some embodiments, the present inventionmonitors fluorescent light emittance and is expected to be highlyaffordable, minimally invasive (requiring only a single injection of asafe dose of nanoparticles), and can be conducted and interpreted in anambulatory setting by a trained emergency medical technician or aprimary care physician, similar to ECG monitoring.

One general aspect of the invention is an imaging agent for detection ofa thrombus in a subject. The agent is composed, in an exemplaryembodiment, of three elements, as follows: i) a fluorescent nano-diamondparticle (NDP-F); ii) a ligand that functionalizes the NDP-F, such as a—COOH, —OH, —NH₂, or —C═O moiety; and iii) a protein attached to theligand. The three can be bonded together in any order and by any type ofchemical bonds, but are typically covalently bonded in the orderdescribed. The objective of the imaging agent is to specifically bindthe agent to a discrete biological target in a human or non-human animalbody.

Yet another aspect of the invention is a diagnostic method for detectionof activated platelets. In some cases, the diagnostic method is based inlarge part on the ability of a species such as a polypeptide (e.g.,bitistatin, BSA, human IgG) to bind to a specific antigen (e.g., PFR)with high affinity and/or avidity. In some embodiments, the polypeptide(e.g., bitistatin) is configured to target the NDP-F to e.g., activatedplatelets, and thus sites of thrombus formation. In some embodiments,the fluorescence of the NDP-F allows non-invasive and relativelyharmless imaging of the location and size of the thrombus, or multiplethrombi. In certain embodiments, the diagnostic method is qualitative,and in other embodiments it is semi-quantitative or quantitative. Insome embodiments, the method comprises: i) administering to a subjectsuspected of having, or potentially having, one or more sites ofthrombus, a detectable amount of the diagnostic agent of the invention,ii) allowing sufficient time for the diagnostic agent to localize to thesite(s) of thrombus, and iii) detecting the diagnostic agent bydetecting fluorescence emission after excitation with a suitableelectromagnetic stimulus (e.g., excitation light, such as emission froma hand held device). It is to be understood that, in some embodiments,in step iii) the act of excitation can be omitted if the fluorescent tagis intrinsically fluorescent in the subject's body. The step ofadministering can be any action that results in introduction of theimaging agent into the systemic blood stream of the subject. It thus maybe, for example and without limitation, via intravenous injection orinfusion.

Yet further, and in accordance with the method described immediatelyabove, the invention includes a method of detecting clots or clotformation in subjects. As with the method described above, this methodcan be considered a method of detecting or imaging clots, clotformation, platelet activation, or pathological zones that form a riskfor clot formation, such as an atherosclerotic vascular plaque orinflammation. The method steps are those described above. For example,in some embodiments, a clot in a subject may be detected byadministering a plurality of nanodiamond particles functionalized with aspecies (e.g., a polypeptide such as bitistatin) which may bind to atleast a portion of a clot.

As those of skill in the art will immediately recognize, the diagnosticand imaging methods of the present invention, by virtue of introductionof a non-natural bio-active substance into a subject's body, do notrelate solely to collection of data regarding a biological event, butinstead relate to physical and physiological changes to the subject'sbody. For example, introduction of the non-naturally occurring imagingagent into the blood stream of a subject physically alters the make-upof the blood stream. In addition, binding of the imaging agent toactivated platelets alters the body's natural ability to interact withthe activated platelets, and thus the clotting cascade. Other physicaland physiological changes upon administration of the imaging agent ofthe invention will be apparent to the skilled artisan.

The present invention also encompasses kits for practicing the methodsof the invention. Broadly speaking, a kit according to the inventionincludes the imaging agent of the invention in packaged form suitablefor distribution, delivery, and/or storage for use in a method of theinvention. In customary fashion, the package is made of a suitablematerial, such as, but not limited to, a cardboard or plastic box andthe like, a metal container and the like, or a foil pouch or the like.In some embodiments, the kit includes sufficient imaging agent in acontainer for a single administration, whereas in other embodiments, thekit includes sufficient imaging agent for two or more administrations.In embodiments where the kit includes sufficient imaging agent for twoor more administrations, the imaging agent can be supplied in a singlecontainer for multiple uses or in two or more containers, eachcontaining sufficient imaging agent for a single use. In some cases, acombination of single-use and multiple-use containers can be included ina kit. In certain embodiments, the kit (regardless of how manycontainers of imaging agent are provided in the kit) can be provided inpackaged combination with one or more reagents or devices foradministration of the imaging agent to a subject. As such, and withoutlimitation, a kit of the invention can include, in packaged combination,the imaging agent with an antiseptic (e.g., ethanol swabs or pads oriodine swabs or pads), one or more syringes, one or more needles adaptedto connect with a syringe, and/or one or more pieces of gauze and/oradhesive to facilitate closure and healing of the site ofadministration.

In some embodiments, a plurality of nanodiamond particles (e.g., NDP-F)bound to a species such as a polypeptide or polynucleotide may beintroduced into a sample suspected of containing an analyte. In certainembodiments, the polypeptide and/or polynucleotide may be selected suchthat, if present, the polypeptide and/or polynucleotide binds to theanalyte.

In some embodiment, if an analyte is present in the sample, the analyte,the species, and/or the nanodiamond particle may bind such that thepresence of the analyte may be detected. In some cases, the detection ofthe analyte comprises quantification of an emission (e.g., a fluorescentemission, a near infrared emission) by the nanodiamond particle. In someembodiments, quantification of the emission comprises quantification ofa relative intensity and/or quantification of a wavelength of theemission. Without wishing to be bound by theory, in some cases, theintensity of the emission may be proportional (e.g., directlyproportional, exponentially proportional, logarithmically proportional)to the amount of analyte present in the sample.

In some embodiments, the plurality of (fluorescent) nanodiamondparticles are administered to a subject. In certain embodiments, theplurality of nanodiamond particles may be administered orally, rectally,vaginally, nasally, or uretherally to the subject. In some cases, theplurality of nanodiamond particles are administered surgically (e.g.,implanted) and/or injected (e.g., into the systemic circulation,intraoccularly, into the spinal system, e.g., via syringe).

In another exemplary embodiment, the plurality of nanodiamond particles(e.g., the plurality of nanodiamond particles comprising a species boundto the nanodiamond particles) may be administered to a subject (e.g.,for the detection of an analyte suspected of being present in thesubject). For example, in some cases, the plurality of nanodiamondparticles comprising the species may be administered to the subject and,upon detection of an emission (e.g., fluorescent emission, near infraredemission) of the nanodiamond particles, demonstrate the presence of ananalyte in the subject. In some cases, the analyte may be at least aportion of a (blood) clot capable of binding to the nanodiamondparticles. In some embodiments, a detection device configured to measureand/or detect a fluorescent emission and/or a near infrared emission maybe applied to the subject (e.g., on or near the skin, at a locationinternal of the subject) such that, if the analyte is present, theemission is detected and/or quantified.

As described above, the methods, devices, and systems described hereinmay be useful for determining and/or quantifying the amount of analytepresent in a sample. In some embodiments, the sample is a fluid. Incertain embodiments, the sample is whole blood. In certain embodiments,the sample is obtained from a subject such as whole blood, plasma,urine, sputum, sweat, and/or other biological fluids. Methods forcollecting such samples are known in the art. In some embodiments, thesample is introduced into a fluidic device (e.g., a fluidic devicecomprising a reservoir comprising a plurality of nanodiamond particles).

In certain embodiments, the sample may be diluted (e.g., prior todetermining and/or quantifying the amount of analyte present in thesample). For example, in certain embodiments, a buffer solution may beadded to the fluidic device comprising the plurality of nanodiamondparticles before, during, and/or after introducing the sample to thefluidic device such that the sample is diluted. In some embodiments, thebuffer solution is contained within a reservoir in fluidic communicationwith one or more components of the fluidic device. The sample may bediluted in a buffer solution prior to, or during, the introduction ofthe plurality of nanodiamond particles to the sample. In someembodiments, an analyte in a sample is readily determinable without anysubsequent process steps. In some cases, the analyte is present in asubject and the nanodiamond particles may be administered to thesubject, as described above.

The term ‘nanodiamond particle’ generally refers to a diamond particlehaving an average cross-sectional dimension of less than 1 micrometer(e.g., less than or equal to 900 nanometers, less than or equal to 800nanometers, less than or equal to 700 nanometers, less than or equal to600 nanometers, less than or equal to 500 nanometers, less than or equalto 400 nanometers, less than or equal to 300 nanometers, less than orequal to 200 nanometers, less than or equal to 100 nanometers, less thanor equal to 90 nanometers, less than or equal to 80 nanometers, lessthan or equal to 70 nanometers, less than or equal to 60 nanometers,less than or equal to 50 nanometers, less than or equal to 40nanometers, less than or equal to 30 nanometers, less than or equal to20 nanometers, or less than or equal to 10 nanometers). In some cases,the nanodiamond particle may have an average cross-sectional dimensionof greater than or equal to 5 nanometers, greater than or equal to 10nanometers, greater than or equal to 20 nanometers, greater than orequal to 30 nanometers, greater than or equal to 40 nanometers, greaterthan or equal to 50 nanometers, greater than or equal to 60 nanometers,greater than or equal to 70 nanometers, greater than or equal to 80nanometers, greater than or equal to 90 nanometers, greater than orequal to 100 nanometers, greater than or equal to 200 nanometers,greater than or equal to 300 nanometers, greater than or equal to 400nanometers, greater than or equal to 500 nanometers, greater than orequal to 600 nanometers, greater than or equal to 700 nanometers,greater than or equal to 800 nanometers, or greater than or equal to 900nanometers. Combinations of the above-referenced ranges are alsopossible (e.g., less than 1 micrometer and greater than or equal to 5nanometers, less than or equal to 700 nanometers and greater than orequal to 100 nanometers). Other ranges are also possible. Those ofordinary skill in the art would be capable of selecting suitable methodsfor determining the average cross-sectional dimension of a nanodiamondbased upon the teachings of this specification.

Without wishing to be bound by theory, in some cases, the nanodiamondparticles described herein may be auto-fluorescent (e.g., thenanodiamond particles emit fluorescent light e.g., after absorption ofelectromagnetic radiation). In some cases, the nanodiamond particles maycomprise one or more atomistic-type defects (e.g., a point defect suchas a nitrogen-vacancy (NV) center, a point defect such as anitrogen-vacancy-nitrogen (NVN) defect, combinations thereof) whichresult in near-infrared fluorescence and/or photoluminescence that maybe detected and/or quantified. Other defects are also possible (e.g.,Si-vacancy defects). In certain embodiments, the nanodiamond particlesfluoresce in response to an applied electromagnetic radiation.

For example, in some embodiments, the nanodiamond particle may beexcited (e.g., by applying electromagnetic radiation having a firstwavelength) such that the nanodiamond particle emits a detectableemission (e.g., an electromagnetic radiation having a second wavelength,different than the first wavelength). In a particular set ofembodiments, if an analyte is present in a sample, the analyte binds tothe nanodiamond particle (e.g., binds to a species bound to thenanodiamond particle) such that an emission from the nanodiamondparticle may be detected and/or quantified. In some cases, detection ofan emission of nanodiamond particles in a subject may indicate that thenanodiamond particles are bound to the suspected analyte. In some suchcases, the emission may be quantified (e.g., to determine the relativeamount of analyte present in the subject).

In another set of embodiments, the sample suspected of containing theanalyte may be added to a fluidic device such that, if present, theanalyte binds to the nanodiamond particles (e.g., to the species boundto the nanodiamond particles) and to a detection region in the fluidicdevice. In some such embodiments, the presence of an emission indicatesthe presence of the analyte in the sample. In some cases, the intensityand/or wavelength of the emission may be quantified.

As described herein, in some embodiments, the systems, devices, andmethods comprise a plurality of nanodiamond particles and a speciesbound to the plurality of nanodiamond particles. Advantageously, thedevices and methods described herein may, in some embodiments, permitthe analysis of analytes from whole blood without additional filteringor separation steps and/or have relatively high sensitivity as comparedto certain existing analyte quantification methods.

As illustrated in FIG. 1A, in some embodiments, device 100 comprises aplurality of nanodiamond particles 110 associated with a species 120(e.g., a species which may bind to an analyte, if present). In someembodiments, the nanodiamond particles are associated with (e.g., boundto) the species via functionalization of the nanodiamond particle. Forexample, in some embodiments, a nanodiamond particle is associated witha species via formation of a bond, such as an ionic bond, a covalentbond, a hydrogen bond, Van der Waals interactions, and the like. Thecovalent bond may be, for example, a carbon-carbon, carbon-oxygen,oxygen-silicon, sulfur-sulfur, phosphorus-nitrogen, carbon-nitrogen,metal-oxygen, or other covalent bond. The hydrogen bond may be, forexample, between hydroxyl, amine, carboxyl, thiol, and/or similarfunctional groups. For example, the species may include a functionalgroup, such as a thiol, aldehyde, ester, carboxylic acid, hydroxyl, andthe like, wherein the functional group forms a bond with the nanodiamondparticle. In some cases, the species may be an electron-rich orelectron-poor moiety wherein interaction between the nanodiamondparticle and the species comprises an electrostatic interaction.

For example, the species may be associated with a functionalizednanodiamond particle comprising a —COOH, —OH, —NH₂, —SH, or —C═Ofunctional group by reacting the functionalized nanodiamond particle andthe species in the presence of a cross-linking agent. Non-limitingexamples of suitable cross-linking agents include carbodiimides such as1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC);amine-reactive compounds such as N-Hydroxysuccinimide ester, imidoester,and hydromethylphosphine; sulfhydryl-reactive compounds such asmaleimide, pyridyl disulfides, and iodoacetyl; aldehyde-reactivecompounds such as hydrazide and alkoxyamine; and photoreactivecross-linking agents such as aryl azides and diazirine. Othercross-linking agents are also possible. Those of ordinary skill in theart would be capable of selecting suitable cross-linking agents basedupon the type of species selected and the teachings of thisspecification.

In some embodiments, the species may bind with a target analyte. In somecases, the species may comprise a biological or a non-biological(chemical) group capable of binding another biological or chemicalmolecule in a sample (e.g., a biological or chemical molecule present onan analyte). For example, the species may include a functional group,such as a thiol, aldehyde, ester, carboxylic acid, hydroxyl, amine,polyethelene glycol and the like, wherein the functional group forms abond with the analyte. In some cases, the species may be anelectron-rich or electron-poor moiety wherein interaction between theanalyte and the species comprises an electrostatic interaction.

In some embodiments, the species and analyte interact via a bindingevent between pairs of biological molecules including proteins, nucleicacids, glycoproteins, carbohydrates, hormones, and the like. Specificexamples include an antibody/peptide pair, an antibody/antigen pair, anantibody fragment/antigen pair, an antibody/antigen fragment pair, anantibody fragment/antigen fragment pair, an antibody/hapten pair, anenzyme/substrate pair, an enzyme/inhibitor pair, an enzyme/cofactorpair, a protein/substrate pair, a nucleic acid/nucleic acid pair, aprotein/nucleic acid pair, a peptide/peptide pair, a protein/proteinpair, a protein/receptor pair, a small molecule/protein pair, aglutathione/GST pair, an anti-GFP/GFP fusion protein pair, a Myc/Maxpair, a maltose/maltose binding protein pair, a carbohydrate/proteinpair, a carbohydrate derivative/protein pair, a metal bindingtag/metal/chelate, a peptide tag/metal ion-metal chelate pair, apeptide/NTA pair, a lectin/carbohydrate pair, a receptor/hormone pair, areceptor/effector pair, a complementary nucleic acid/nucleic acid pair,a ligand/cell surface receptor pair, a virus/ligand pair, a ProteinA/antibody pair, a Protein G/antibody pair, a Protein L/antibody pair,an Fc receptor/antibody pair, a biotin/avidin pair, abiotin/streptavidin pair, a drug/target pair, a zinc finger/nucleic acidpair, a small molecule/peptide pair, a small molecule/protein pair, asmall molecule/target pair, a carbohydrate/protein pair such asmaltose/MBP (maltose binding protein), a small molecule/target pair, anda metal ion/chelating agent pair. Specific non-limiting examples ofspecies include peptides, proteins, DNA, RNA, and PNA. Other species andbinding pairs are also possible. In an exemplary embodiment, the speciesis an antibody (e.g., an antibody to a target analyte).

In an exemplary embodiment, the species and analyte interact via anantibody/antigen pair binding event.

In another exemplary embodiments, the species and analyte interact via aprotein/receptor pair binding event. For example, the species maycomprise a protein such as disintegrin (e.g., Bitistatin) and theanalyte may comprise a receptor molecule such as a fibrinogen receptor.

In some embodiments, the species and the analyte interact via a bindingevent between pairs of biological molecules including proteins, nucleicacids, glycoproteins, carbohydrates, hormones, or the like.

In some embodiments, the species is selected from the group consistingof (poly)peptides, (poly)nucleotides, and ligands. In an exemplaryembodiment, the species is a polypeptide, such as a protein, anantibody, and/or an antigen. For example, in some embodiments, theantibody is an immunoglobulin such as IgA, IgG, IgM, IgE, or the like,In another exemplary embodiment, the species is a (poly)nucleotide(e.g., an oligonucleotide), such as DNA or RNA. In yet another exemplaryembodiment, the species is a disintegrin such as albolabrin, applagin,barbourin, batroxostatin, bitistatin, obtustatin, schistatin,echistatin, elegantin, eristicophin, flavoridin, halysin, kistrin,mojastin, rubistatin, tergeminin, salmosin or triflavin. In someembodiments, the species may comprise a protein such as serum albumin(e.g., bovine serum albumin).

Examples of suitable nanodiamond particles functionalized with a species(e.g., Bitistatin) are discussed in more detail in U.S. ProvisionalPatent Application No. 62/383,657, filed Sep. 6, 2016, entitled“Engineering and Utility of Fluorescent Nanodiamond Particles (NDP-F)for Diagnostics and Treatment of Blood Clots in Human and VeterinaryMedicine,” which is incorporated herein by reference in its entirety.Other functionalization methods are also possible.

Non-limiting examples of analytes that may be detected and/or quantifiedinclude a biological compound, a drug, a macromolecule, a salt, anelectrolyte, an enzyme, a nucleic acid (e.g., a (poly)nucleotide), acarbohydrate, a (poly)peptide, a protein, a lipid, a phosphate, asulfonate, a virus, a pathogen, a bacterium, a fungus, an oxidant, areductant, a toxin, a surfactant, and combinations thereof.

In an exemplary set of embodiments, the analyte is a virus (e.g., ebola,Marburg, Bundibugyo, sudan, junin, lassa, MERS, small pox, Zika,pertussis, rubella, rubeola). In some embodiments, the analyte is abacterial toxin (e.g., anthrax). In some embodiments the analyte is abiological entity associated with a particular parasite and/or fungus.Advantageously, the systems and methods described herein may be usefulfor the detection and/or quantification of viruses such as ebola.

In yet another exemplary set of embodiments, the analyte is a receptormolecule (e.g., a fibrinogen receptor). Advantageously, the systems andmethods described herein may be useful for the detection of blood clots.

In some cases, the analyte may comprise a marker/antigen for aparticular disease or condition. For example, in some cases, the analytemay be a marker/antigen associated with blood clots, traumatic braininjury, bone diseases (e.g., osteroporosis, osteoarthrosis),inflammation and/or (auto)immune diseases (e.g., Crohn, psoriasis)ulcers, cardiac ischemia and stroke, atherosclerosis, muscle diseases,Alzheimer's/Parkinson's, tumors and tumor metastasis, or others. Forexample, the systems and methods described herein may be useful for thedetection and/or diagnosis in a subject of blood clots, traumatic braininjury, inflammation and/or (auto)immune diseases (e.g., Crohn's,psoriasis), ulcers, cardiac ischemia and stroke, atherosclerosis, musclediseases, Alzheimer's/Parkinson's, tumors and tumor metastasis, orothers.

In some embodiments, the plurality of nanodiamond particles and thespecies associated with the nanodiamond particles are present (e.g., ina reservoir) and/or introduced into a fluidic device. For example, asillustrated in FIG. 1B, fluidic device 102 comprises a sample inlet 130and a reservoir 140 in fluidic communication with sample inlet 130. Incertain embodiments, reservoir 140 comprises a plurality of(fluorescent) nanodiamond particles 110 (and species 120 associated withnanodiamond particle 110).

In certain embodiments, a sample may be introduced to the sample inletsuch that the sample flows into the reservoir and the sample interactswith the plurality of nanodiamond particles and species. In someembodiments, an analyte, if present in the sample, binds to the species.

In some cases, detection region 150 may be positioned downstream of,and/or in fluidic communication with, reservoir 140.

In certain embodiments, a detector 160 may be positioned proximatedetection region 150. The detector, in some cases, may be configured toquantify an emission (e.g., an intensity of the emission, a wavelengthof the emission) at the detection region. In some cases, the emissionmay be fluorescent and/or near-infrared. For example, the analyte, ifpresent, may bind to the species. In some such cases, the analyte maybind to a second species (e.g., an antibody) associated with (e.g.,bound to) the detection region. In some embodiments, the analyte boundto the second species and the first species (associated with thenanodiamond particles) may be detected and/or quantified by measuring(e.g., via the detector) the emission of the nanodiamond particles.

In some embodiments, a detector may be positioned proximate a region ofa subject suspected of containing an analyte and/or a clot. For example,the plurality of (fluorescent) nanodiamond particles functionalized witha species may be administered to a subject, and the detector may bepositioned proximate the subject such that any nanodiamond particlesbound to the analyte and/or clot may be detected (e.g., via an emissionof the nanodiamond particles).

Any suitable detector may be used with the devices and methods describedherein. For example, in some embodiments, the detector may be an opticaldetector (e.g., fluorescence detectors, visible light and/or UVdetectors, near infrared detectors, microscopes).

In some embodiments, the emission is a fluorescent emission. In certainembodiments, the wavelength of the emission is greater than or equal to250 nm, greater than or equal to 300 nm, greater than or equal to 350nm, greater than or equal to 400 nm, greater than or equal to 450 nm,greater than or equal to 500 nm, greater than or equal to 550 nm,greater than or equal to 600 nm, or greater than or equal to 650 nm. Incertain embodiments, the wavelength of the emission is less than orequal to 700 nm, less than or equal to 650 nm, less than or equal to 600nm, less than or equal to 550 nm, less than or equal to 500 nm, lessthan or equal to 450 nm, less than or equal to 400 nm, less than orequal to 350 nm, or less than or equal to 300 nm. Combinations of theabove-referenced ranges are also possible (e.g., greater than or equalto 250 nm and less than or equal to 700 nm). Other ranges are alsopossible.

In certain embodiments, the emission is a near infrared emission. Insome embodiments, the wavelength of the emission is greater than 700 nm,greater than or equal to 750 nm, greater than or equal to 800 nm,greater than or equal to 850 nm, greater than or equal to 900 nm, orgreater than or equal to 950 nm. In certain embodiments, the wavelengthof the emission is less than or equal to 1000 nm, less than or equal to950 nm, less than or equal to 900 nm, less than or equal to 850 nm, lessthan or equal to 800 nm, or less than or equal to 750 nm. Combinationsof the above-referenced ranges are also possible (e.g., greater than 700nm and less than or equal to 1000 nm). Other ranges are also possible.

In some embodiments, the nanodiamond particle may emit a fluorescentand/or near infrared emission upon excitation by electromagneticradiation having a particular wavelength. For example, in someembodiments, the nanodiamond particle may be exposed to electromagneticradiation having a wavelength of greater than or equal to 250 nm,greater than or equal to 300 nm, greater than or equal to 350 nm,greater than or equal to 400 nm, greater than or equal to 450 nm,greater than or equal to 500 nm, greater than or equal to 550 nm,greater than or equal to 600 nm, greater than or equal to 650 nm,greater than or equal to 700 nm, greater than or equal to 750 nm,greater than or equal to 800 nm, greater than or equal to 850 nm,greater than or equal to 900 nm, or greater than or equal to 950 nm(e.g., such that the nanodiamond particle emits a fluorescent emissionand/or near infrared emission in one of the above-referenced ranges). Incertain embodiments, the nanodiamond particle may be exposed toelectromagnetic radiation having a wavelength of less than or equal to1000 nm, less than or equal to 950 nm, less than or equal to 900 nm,less than or equal to 850 nm, less than or equal to 800 nm, or less thanor equal to 750 nm, less than or equal to 700 nm, less than or equal to650 nm, less than or equal to 600 nm, less than or equal to 550 nm, lessthan or equal to 500 nm, less than or equal to 450 nm, less than orequal to 400 nm, less than or equal to 350 nm, or less than or equal to300 nm. Combinations of the above-referenced ranges are also possible(e.g., greater than or equal to 250 nm and less than or equal to 1000nm, greater than or equal to 550 nm and less than or equal to 650 nm).Other ranges are also possible.

While much of the description herein is in the context of (fluorescent)nanodiamond particles, those of ordinary skill in the art wouldunderstand, based upon the teachings of this specification, that otherparticles are also possible. For example, in some embodiments, thedevice may comprise a particle such as a nanoparticle (e.g., a silicananoparticle, a sapphire nanoparticle, a garnet nanoparticle, a rubynanoparticle) having an emission in one of the above referenced rangesassociated with a species (e.g., a species capable of binding to one ormore target analytes). In some cases, the particle may beautofluorescent. In other cases, the particle may be functionalized with(e.g., associated with) a fluorescent molecule.

In some embodiments, the fluidic device (e.g., comprising a sample inletand a reservoir comprising a plurality of nanodiamond particles and aspecies associated with the nanodiamond particles) comprises a lateralflow assay configuration (e.g., in a lateral flow device). Those ofordinary skill in the art would understand, based upon the teachings ofthis specification, how to incorporate a plurality of nanodiamondparticles and a species associated (e.g., bound) to the nanodiamondparticles into a lateral flow assay device. For example, as illustratedschematically in FIG. 2A, in an exemplary embodiment, system 200comprises a lateral flow assay format. In certain embodiments, system200 comprises reservoir 210 comprising a plurality of fluorescentnanodiamond particles 220 bound to a first species. Downstream ofreservoir 210, in certain embodiments, is a second reservoir 230comprising a second species (e.g., a first antibody to a target analyte)and a third reservoir 240 comprising a third species (e.g., a secondantibody capable of binding to the antibody to the target analyte). Incertain embodiments, the first species and the second species are thesame.

Now referring to FIG. 2B, a sample suspected of containing an analyte250 may be introduced into system 200 (e.g., such that the sample flowsand interacts with reservoir 210). In some embodiments, at least aportion of analyte 250 binds to the species on plurality of fluorescentnanodiamond particles 220. As illustrated in FIG. 2C, as the samplesuspected of containing analyte 250 flows along the system, at least aportion of the analytes 250 (e.g., now bound to the species and/orplurality of fluorescent nanodiamond particles 220) bind to the secondspecies in reservoir 230. In some cases, at least a portion of pluralityof fluorescent nanodiamond particles 220 not bound to the analyte may becaptured in reservoir 240 (e.g., where the third species is capable ofbinding to the first species).

In some cases, the devices and systems herein may be multiplexed. Thatis to say, in some embodiments, the devices may comprise two or more,three or more, four or more, or five or more fluidic components and/orreservoirs comprising a plurality of nanodiamond particles. In some suchembodiments, more than one analyte may be detected, if present in thesample, in a single device. In certain embodiments, one or more analytesmay be detected in a device comprising a plurality of fluidic componentsand/or reservoirs comprising a plurality of nanodiamond particles.

In an exemplary embodiment, a sample suspected of containing an analytemay be introduced into a fluidic channel of a fluidic device, exposingthe sample to a species bound to a plurality of fluorescent nanodiamondparticles such that the analyte, if present, binds to at least a portionof the species bound to the plurality of fluorescent nanodiamondparticles. In some embodiments, any fluorescent nanodiamond particlesand species not bound to the analyte may be removed and a fluorescenceemission of the plurality of fluorescent nanodiamond particles bound tothe analyte may be quantified. As described herein, in some cases, theamount of analyte present in the sample may be correlated with theintensity of the fluorescence emission.

In another exemplary embodiment, a system comprises a sample inlet, areservoir in fluidic communication with the sample inlet, the reservoircomprising a plurality of fluorescent nanodiamond particles, a pluralityof a first species bound to the plurality of fluorescent nanodiamondparticles, and a detection region in fluidic communication with thereservoir, the detection region comprising a plurality of a secondspecies bound to the detection region. In some cases, the detector maybe configured to quantify a fluorescent emission at the detection regionand/or configured to quantify an infrared signal at the detectionregion.

In yet another exemplary embodiment, a fluidic device comprises a sampleinlet, a reservoir in fluidic communication with the sample inlet, thereservoir comprising a plurality of fluorescent nanodiamond particles, aplurality of a first species bound to the plurality of fluorescentnanodiamond particles, and a detection region in fluidic communicationwith the reservoir, the detection region comprising a plurality of asecond species bound to the detection region. In some embodiments, thefluidic device may further comprise a control region in fluidiccommunication with the detection region, the control region comprising aplurality of a third species bound to the control region. In some suchembodiments, the third species may be the same or different as the firstspecies and/or the second species. For example, the first species boundto the plurality of fluorescent nanodiamond particles may be a firstantibody (e.g., capable of binding selectively to the target analyte),the second species may be the first antibody or may be a second antibodydifferent than the first antibody (e.g., capable of binding selectivelyto the target analyte), and the third species may be an anti-antibody tothe first antibody. The control region may provide an indication, if anemission is present, that the system is working properly (e.g., theplurality of nanodiamond particles were properly featured and introducedinto the sample).

In some embodiments, the fluidic device comprises an absorbent material.In some embodiments, an absorbent region comprising the absorbentmaterial is positioned downstream of, and in fluidic communication with,the detection region and/or the control region. In some cases, theabsorbent material is associated with one or more components (e.g., thereservoir, the detection region) of the fluidic device. In some cases,the absorbent material may at least partially drive the flow of thesample in the fluidic device (e.g., wicking). In other embodiments,capillary action may at least partially drive the flow of the sample inthe fluidic device.

Non-limiting examples of suitable absorbent materials include solidmaterials, porous materials, particles, powders, and gels. In someembodiments, the absorbent material may comprise fabric, cellulose,cotton, and/or a polymer. Those of ordinary skill in the art would becapable of selecting suitable absorbent materials based upon theteachings of this specification.

In another exemplary embodiment, the methods described herein compriseadministering, to a subject suspected of having a particular analyte(e.g., present in the bloodstream), a plurality of fluorescentnanodiamond particles bound to a species such that the species may bindto the analyte, if present, and detecting a fluorescent and/or nearinfrared emission of the plurality of fluorescent nanodiamond particlescomprising the species bound to, if present, the analyte. In some cases,detecting a fluorescent emission indicates the presence of a blood clotin the subject. In certain embodiments, detecting a fluorescent emissionindicates the presence of a virus (e.g., ebola, Marburg, Bundibugyo,sudan, junin, lassa, MERS, etc.) in the subject.

As should be evident, in some embodiments, the present invention alsoprovides a diagnostic agent for detection or imaging of thromboticevents in a human or non-human animal, where the agent comprises afluorescent nanodiamond particle chemically bonded to disintegrinBitistatin (Bt). In certain embodiments, the fluorescent nanodiamondparticle and the Bt are covalently bonded. The diagnostic agent may befluorescent as a result of an intrinsic property of the nanodiamondparticle. In some embodiments, the diagnostic agent emits a detectableelectromagnetic signal when excited by an electromagnetic source.

Additional exemplary embodiments relate to a method for diagnosis orprognosis of a thrombo-embolic event. In these embodiments, the methodcomprises: administering to a subject suspected of having suffered fromor suspected of being at risk of, a thrombo-embolic event, adiagnostically effective amount of the diagnostic agent of theinvention; allowing sufficient time for the diagnostic agent to localizeto the site(s) of thrombus; and detecting the diagnostic agent bydetecting fluorescence emission of the diagnostic agent. The method canbe practiced as a method of detection of activated platelets and/or amethod of detecting clots or clot formation in subjects.

Another exemplary embodiment of the invention relates to a kit. The kitmay include the diagnostic agent of the invention in packaged formsuitable for distribution, delivery, and/or storage for use in adiagnostic method (e.g., a diagnostic method for detection of athrombus). The packaged form may include a suitable material fordistribution, delivery, and/or storage of the diagnostic agent. Incertain embodiments, the kit further comprises, in packaged combination,one or more reagents or devices for administration of the diagnosticagent of the invention to a subject. The kit can also include a devicethat emits excitation energy for the diagnostic agent, and preferablyfurther includes a detector for detection of emission response from thediagnostic agent. In a particular exemplary embodiment, the device(e.g., a device that emits excitation energy, a detector for detectionof emission response) is a hand-held device.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the practice of the presentinvention without departing from the scope or spirit of the invention.Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention. It is intended that the specification and examples beconsidered as exemplary only, with a true scope and spirit of theinvention being indicated by the following claims.

A “subject” or a “patient” refers to any mammal (e.g., a human), forexample, a mammal that may be susceptible to a disease or bodilycondition. Examples of subjects or patients include a human, a non-humanprimate, a cow, a horse, a pig, a sheep, a goat, a dog, a cat or arodent such as a mouse, a rat, a hamster, or a guinea pig. Generally,the invention is directed toward use with humans. A patient may be asubject diagnosed with a certain disease or bodily condition orotherwise known to have a disease or bodily condition. In someembodiments, a patient may be diagnosed as, or known to be, at risk ofdeveloping a disease or bodily condition. In other embodiments, apatient may be suspected of having or developing a disease or bodilycondition, e.g., based on various clinical factors and/or other data. Insome cases, the patient may be diagnosed with having or developing aparticular disease or bodily condition after the detection and/orquantification of an analyte (e.g., a virus, an antigen, an antibody) ina sample obtained from the patient. In some embodiments, a subject maydemonstrate health benefits, e.g., upon administration of thefluorescent nanodiamond particles.

As used herein, a “fluid” is given its ordinary meaning, i.e., a liquidor a gas. A fluid cannot maintain a defined shape and will flow duringan observable time frame to fill the container in which it is put. Thus,the fluid may have any suitable viscosity that permits flow. If two ormore fluids are present, each fluid may be independently selected amongessentially any fluids (liquids, gases, and the like) by those ofordinary skill in the art.

The following examples are intended to illustrate certain embodiments ofthe present invention, but do not exemplify the full scope of theinvention.

EXAMPLES Prophetic Example 1

The following example demonstrates an exemplary functionalizedfluorescent nanodiamond particle with a species (e.g., for binding toanti-human IgG-AP).

FIG. 3 illustrates the conjugation of a fluorescent nanodiamond particle(F-NDP), functionalized with a carboxylic acid group, and bound to aspecies (e.g., Bitistatin). Such functionalized F-NDPs can be used, forexample, to image and detect the presence of blood clots using externalscanners (e.g., detectors for UV and/or NIR emissions).

Example 2

The following example demonstrates the incorporation of fluorescentnanodiamond particles into an exemplary system such as a lateral flowassay format.

FIG. 4 shows the loading of three exemplary systems (system 400, system410, and system 420), each with a reservoir of fluorescent nanodiamondparticles (shown as the dot in each system) placed on a nitrocellulosesubstrate. Upon introduction of water to system 400, water with 1% Tween20 to system 410, and 10 mM sodium phosphate with 1% Tween 20 to system420, flow of the fluorescent nanodiamond particles along the substrateof each device was observed (system 402, system 412, and system 422respectively).

Example 3

The following example demonstrates the use of fluorescent nanodiamondparticles (F-NDP) conjugated with a species (e.g., human IgG) for thedetection of an analyte (e.g., anti-human IgG).

FIG. 5 shows a plot of semi-ELISA for detection of human IgG coupled tothe F-NDP NV (700 nm). F-NDP were coupled to human IgG, or BSA(control), which were used in concentrations as indicated, per 1 mg ofF-NDP. 0.2 mg of each F-NDP-IgG or F-NDP-BSA sample were used forsemi-ELISA, which was performed on a U-shape bottom 96-well plate. Theplate was blocked overnight with 3% BSA in PBST. F-NDP-IgG were blockedwith 3% BSA in PBST by incubation for 1 hour at 37° C. Blocking agentwas removed by centrifugation (1,000 g) at room temperature and washed2× with PBST. Goat anti-human IgG AP-conjugated (Sigma Inc.), diluted1:5000, was added and incubated for 1 hr. as above. Final washing wasperformed as above, and p-nitrophenyl phosphate substrate (pNPP) toalkaline phosphatase (AP) was added. Color was developed for 30 min, andNDP samples on plate were centrifuged. Supernatant was transferred to aflat bottom 96-well plate and read using an ELISA plate reader under 405nm wave length. Error bars represent four repeats in semi-ELISA from thesame samples.

Example 4

The following example demonstrates the use of F-NDPs functionalized witha species (e.g., Bitistatin (Bt)) for the detection of blood clots. FIG.6 shows the accumulation number of F-NDP-Bt in carotid arteries in ratswith generated clots. Carotid arteries with generated clots or not(control) were dissected from rats at the end of the experiments.Vessels were solubilized with 12 N HCl by overnight incubation at 60° C.in a ratio of 100 mg per ml. The resulting solution was diluted 10× withwater and centrifuged using 14,000 rpm at room temperature andre-suspended in the same volume of water. F-NDP-Bt were counted using ahemocytometer with fluorescence microscope (40× objective, TRITCwavelengths). Presented numbers show total accumulated particles. Errorbars represent standard deviation from three rats (clotvalue=2.8±0.38×10⁷; control value=6.6±4.7×10⁵)

FIG. 7A is a scanning confocal microscope (SCM) image of carotidarteries from rats with a generated clot. Generation of a clot wasperformed using ferric chloride. F-NDP-Bt suspension was injectedlocally, close to the clot. Rats were euthanized, and carotid arterieswith clots were dissected. Imaging was performed using SCM. Wavelengthsused for measurement: excitation Cy5.5 BkG (580-610 nm), emission Cy5.5(695-770 nm). For background subtraction: excitation GFP (445-490 nm),emission Cy5.5 (695-770 nm). NIR detection of F-NDP-Bit is indicated bythe white arrows.

FIG. 7B shows a scanning confocal microscope image of carotid arteriesfrom rats with generated clot. Generation of a clot was performed usingferric chloride. F-NDP-Bt suspension was systemically injected to theanimal tail. The rat was euthanized, and the carotid artery with clotswas dissected. Imaging was performed using SCM. Wavelengths used formeasurement: excitation Cy5.5 BkG (580-610 nm), emission Cy5.5 (695-770nm). For background subtraction: excitation GFP (445-490 nm), emissionCy5.5 (695-770 nm). NIR detection is indicated by white arrows.

FIG. 8 shows images of tissue suspensions of clots treated or not withF-NDP-Bt, injected systematically to the femoral vein close (locally) tothe clot generated in carotid artery. Tissues of the clot were manuallyhomogenized in the presence of RIPA buffer (Triton X-100 based) in ratioone vein for 100 ml. Suspension of the lysate was applied on glass slideand immediately analyzed under the microscope. Magnification 100×.

Example 5

The following example demonstrates the functionalization of F-NDP (i.e.,NDP-F) with a polypeptide (e.g., Bitistatin (Bt)) and the detection ofone or more analytes and/or blood clots.

The methodology of coupling proteins/peptides to carboxyl-functionalizedNDP-F is described herein. Preservation of the active domainsresponsible for the biological action of the coupled proteins/peptidesremains challenging, and is generally considered to be a trial and errorendeavor. A major innovation of the present invention is thedemonstration of a concentration-dependent association of the engineeredNDP-F-Bt agent to purified PFR. The selection of Bt was based on thehigh selectivity/specificity of Bt to the PFR, concomitant withnegligible interactions with other RGD-dependent integrins, such asreceptors for vitronectin (avb3) and fibronectin (a5b1). The presentstrategy to utilize Bt for clot imaging differs significantly fromprevious studies aimed at demonstrating the utility of Tc99-Bitistatinto map blood clots in vivo. The present imaging strategy is based on theinnate near infrared (NIR) fluorescence emitted upon excitation of theNDP-F, thereby eliminating high radioactivity exposure required by otherimaging techniques. It is also envisioned that coupling Bt (or otherpolypeptides) to a nanoparticle will be beneficial for the extension ofthe lifetime of the Bt at the site of its biological target.

Fluorescent nano-diamond particles (NDP-F or FNDP) functionalized bycarboxyl groups (—COOH) were purchased from Adamas Nanotechnologies,Inc. (Raleigh, N.C.). Size distribution analysis revealed the peak ofthe diameter of the NDP was 734.5 (±223.6, SD) nm. Optimization offluorescence for the NDPs was performed by 2D screening of excitationvs. emission wavelengths (FIG. 9). Two areas of optimal fluorescencewere established for NDPs, which should be useful for application inmedical imaging (circled on FIG. 9). Detailed screening revealed twooptimal correlations of excitation vs. emission wavelengths: 480 nm vs.520 nm, and 565 nm vs. 700 nm. The first correlation represents thetypical green fluorescence, whereas the second is characterized by thelong Stokes shift of fluorescence with near IR emission. The near IRemission is very useful for detection in vivo because it is in theoptical therapeutic window of autofluorescence of factors present inhuman and non-human animal non-invasive imaging environments (e.g.,water, hemoglobin, oxyhemoglobin, melanin).

The NDP-Fs were found to be resistant to photobleaching. Exposure of theslides containing NDPs to intense fluorescence light resulted in nochanges in the intensity of their fluorescence in the time points up to5 hours (data not shown).

Bitistatin is generally derived from snake venom and belongs to thedisintegrin family of proteins. It has previously been investigated as apotential reagent for detection of deep venous thrombosis (DVT) usingradioactive tags. This RGD-disintegrin showed desirable parameters fordetection of DVT when compared with other snake venom disintegrins, suchas kistrin and barbourin. Therefore, this fibrinogen receptor-bindingligand was selected for coupling to NDP-Fs for the purpose of detectingactivated platelets (or their aggregates) in clots present in the venouscirculation (vein thrombus).

Bitistatin is generally an 83-amino acid polypeptide, which originallywas isolated from venom of Bitis arietants. This naturally occurringpolypeptide was purified from the same snake venom using methodologydeveloped in the laboratory for purification of other snake venomdisintegrins (Marcinkiewicz, Cezary, et al. “EC3, a novel heterodimericdisintegrin from Echis carinatus venom, inhibits α4 and α5 integrins inan RGD-independent manner.” Journal of Biological Chemistry 274.18(1999): 12468-12473; incorporated herein by reference). Briefly, thismethod includes two steps of reverse phase HPLC with application of Ciscolumn and a linear gradient of acetonitrile as a protein elution agent.Purity of the obtained Bt was tested by SDS-PAGE and quantified bydigitization of bands on standard protein gels by Coomassie bluestaining. The content of Bt was estimated at over 98% in the finalprotein preparation. This preparation was suspended in PBS for couplingto NDPs.

The active site of Bt was characterized and found to include an RGDmotif, which is important for ligand binding to certain integrins, suchas αIIbβ3, αvβ3, and a5β1. Bt is quite selective in that it has beenshown to bind only the PFR, αIIbβ3 integrin, which is exclusivelyexpressed on circulating platelets. The aspartic acid in the RGDsequence was recognized by site-directed mutagenesis of recombinantproteins and short peptide synthesis as the most essential amino acidfor disintegrin binding to the fibrinogen receptor. It was hypothesizedby the inventors that coupling of Bt to NDPs that were functionalized byan attached amine group could affect the activity of disintegrin byengagement with carboxyl groups present in the side chain of theaspartic acid. Therefore, NDPs functionalized by a carboxyl group wereselected to attach the Bt on NDPs surfaces.

Coupling of carboxyl (—COOH) groups present on NDPs to NH₂ groupspresent on Bt was performed using a standard protocol developedpreviously (Grabarek and Gergely, 1990). This method was based on theapplication of the cross-linker1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), whichactivates carboxyl groups to bind amine groups by forming anO-acylisourea intermediate that quickly reacts with an amino group toform an amide bond. O-acylisourea generally undergoes quick hydrolysis.Therefore, to increase the efficiency of coupling,sulfo-N-hydroxysuccinimide (sulfo-NHS) was added to the reaction mixtureto generate an amine-reactive sulfo-NHS ester.

BSA was coupled in parallel to the control NDPs.

A detailed description of an exemplary suitable coupling procedurefollows: Two samples of NDP, each containing 2 mg in 1.8 ml of 0.1M MES,0.5M NaCl, pH 6.0, were prepared. Sample A contained 1 mg Bt per 1 mgNDPs. Sample B contained 1 mg BSA per 1 mg NDPs, and served as acontrol.

To each sample, 0.72 mg EDC was added and the mixture incubated at roomtemperature (about 21° C. to 25° C.). with gentle rotating for 15minutes. After incubation, 2.5 μl of 2-mercaptoethanol (to give a finalconcentration of 20 mM) was added to quench the EDC and stop thecarboxyl group activation.

The activated NDPs were transferred to 1.5 ml centrifuge tubes andcentrifuged at 10,000×g for 5 minutes at room temperature. The NDPpellets were washed twice with 1 ml PBS and suspended in coupling buffer(PBS). One milligram of either Bt or BSA in 1 ml of PBS was added toeach sample tube. The tubes were incubated for two hours at roomtemperature with gentle rotating. Ten μl of 1M Hydroxylamine-HCl (350 mgin 5 ml PBS) was added to each tube for quenching (final concentrationof Hydroxylamine-HCl should be about 10 mM).

The sample tubes were centrifuged at 10,000×g for 5 minutes at roomtemperature. The supernatants were saved for protein concentrationdetermination to check coupling efficiency. The NDP-F-Bt particles werewashed three times with 1 ml PBS each time. The washed particles weresuspended in a volume of PBS to achieve a concentration of 1 mg/ml.

Coupling efficiency was evaluated using immune detection of Bt on thesurface of NDPs (NDP-Bt). For ease of reference in this document, thedetection method is called semi-ELISA. Broadly speaking, polyclonalantibody against Bt was developed commercially in rabbits(Chemicon/Millipore Inc.) using purified native polypeptide as theantigen. The saturated concentration of Bt was estimated at 1 mg per 1mg NDPs. A representative plot from three independent experiments ispresented in FIG. 10.

An exemplary suitable assay for determining coupling efficiency follows:Using a 96-well plate having a U-shaped bottom and that has previouslybeen blocked by incubation overnight at 4° C. with 5% BSA in PBS, 200 μlof an NDP-F-Bt or NDP-F-BSA solution (1 mg/ml NDP in PBS) was eachapplied to a separate well. The plate was centrifuged for 10 minutes at1,000×g. The supernatant was removed and the plate gently shaken byvortexing to disperse and re-suspend the pellet of NDP-F.

200 μl of 10% goat serum in PBST (PBS+0.05% Tween-20) was added andmixed a few times by intensive aspiration and releasing from a pipettip. The mixture was incubated for one hour at 37° C. with slowagitation. The mixture was centrifuged as above and washed three timeswith 200 μl of PBST. After each centrifugation, the pellet was dispersedby vortexing. After the final wash, 100 μl of anti-bitistatin (5 μg/mlin PBST) was added to the wells and incubated for one hour at roomtemperature. The plate was then washed three times with PBST asdescribed above. To each pellet, 100 μl of a 1:2000 dilution of goatanti-rabbit IgG AP conjugate (from Sigma) in PBST was added to the wellsand the mixture incubated for one hour at 37° C. The mixture wascentrifuged at 1,000×g for 10 minutes, and the pellet was washed threetimes with PBST as above.

150 μl of the AP substrate pNPP (from Sigma) was added and color wasdeveloped for approximately 30 minutes (until the intensity of theyellow color was visible) at room temperature with gentle agitation. Thereaction was blocked by adding 100 μl of 3 M NaOH (this step isoptional). The plate was then centrifuged and 100 μl of the supernatantwas transferred to a 96-well plate (flat bottom). The absorbance of thesupernatant was measured at 405 nm using an ELISA plate reader.

The activity of NDP-Bt was tested using PFR purified from platelets,αIIbβ3 integrin (Millipore Inc.). Two types of assays were applied toconfirm the presence of disintegrin on NDP-Bt, including binding ofsoluble PFR to NDP-F-Bt and adhesion of NDP-F-Bt to fibrinogen receptorimmobilized on the surface (plastic or glass). Binding of soluble PFRwas determined on the basis of the functional “sandwich” ELISA, which isreferred to herein as a functional semi-ELISA. In this case,immune-detection of αIIbβ3 integrin bound to the NDP-Bt was performedusing a polyclonal antibody against the αIIβ integrin subunit (purchasedfrom Santa Cruz Inc.).

An exemplary suitable protocol for a functional semi-ELISA for detectingbinding of fibrinogen receptor to NDP-F-Bt follows:

Using a 96-well plate having a U-shaped bottom and that has previouslybeen blocked by incubation overnight at 4° C. with 5% BSA in PBS, 200 μlof an NDP-F-Bt or NDP-F-BSA suspension (1 mg/ml NDP in PBS) was eachapplied to a separate well. The plate was centrifuged for 10 minutes at1,000×g. The supernatant was removed and the plate gently shaken byvortexing to disperse the pellet of NDP-F. 200 μl of 3% BSA PBST(PBS+0.05% Tween-20) was added and the composition was mixed a few timesby intensive aspiration and releasing from a pipet tip. The mixture wasincubated for one hour at 37° C. with slow agitation. Alternatively, theblocking step may be performed in a tube before application on the96-well plate. The mixture was centrifuged as above and washed threetimes with 200 μl of PBST. After each centrifugation, the pellet wasdispersed by vortexing.

Fibrinogen receptor was then added at the desired concentrations (i.e.,the amounts required to generate a dose-response); see FIG. 11 (in 200μl of HBSS containing physiological concentrations of Ca²⁺ and Mg²⁺ andincubated for one hour at 37° C. The plate was then washed three timeswith PBST as above. Next, 100 μl of anti-fibrinogen receptor (2 μg/mlfrom Santa Cruz Inc.) polyclonal antibody in PBST was added to the wellsand incubated for one hour at room temperature. The plate was washedthree times with PBST as above. To the pellets, 100 μl of a 1:3000dilution of goat anti-rabbit IgG AP conjugate (from Sigma) in PBST wasadded to the wells and incubated for one hour at 37° C. The mixture wascentrifuged at 1,000×g for 10 minutes, and the pellets washed threetimes with PBST as above.

150 μl of the AP substrate pNPP (from Sigma) was added and color wasdeveloped for approximately 30 minutes at room temperature with gentleagitation. The reaction was blocked by adding 100 μl of 3 M NaOH (thisstep is optional). The plate was centrifuged and 100 μl of supernatantwas transferred to the well of a 96-well plate (flat bottom). Theabsorbance of the supernatant was measured at 405 nm using an ELISAplate reader.

As can be seen from FIG. 11, the PFR bound to the NDP-Bt in adose-dependent manner, whereas the control NDP-BSA were not active inthis assay.

Adhesion of NDP-Bt to immobilized fibrinogen receptor was performed intwo formats based on the estimation of the fluorescence of attachedNDPs. First, integrin was immobilized on a 96-well plate and adheredNDPs were detected using a fluorescence plate reader. The resultsobtained showed linear progression of the adhesion of NDP-Bt toincreased concentration of immobilized purified receptor (FIG. 12).Adhesion of NDP-Bt was also monitored under fluorescence microscopy. Thenumber of adhered NDPs was quantified using computer software (FIG. 13).Representative images of adhered NDP-Bt are presented in FIG. 14.

To further characterize the effects of the FNDP, fluorescent images weretaken by IVIS and confocal microscopy of carotid artery clots aftertreatment with FNDP via external carotid artery infusion. The resultsare shown in FIG. 15. In two independent experiments, infusion of FNDPvia the external carotid artery commenced 3-5 minutes after FeCl₃application and continued over 15 minutes (5 minutes beyond the end ofFeCl₃ infusion). The FNDP solution consisted of 1.5 ml of PBS where 5mg/ml of FNDP was suspended (after vortexing of the solution). Thisroute of infusion was selected so as to avoid possible “first pass”elimination of the particles by peripheral organs. Following completionof FNDP infusion, the rat was euthanized and subjected to imaging byIVIS and/or fluorescent microscopy. FIGS. 15A-D show imaging offluorescence that was performed on an IVIS scanner designed for wholeanimal imaging using a 580-610 nm excitation and a 695-770 nm emissionpassband with a 2 second exposure. Auto-fluorescence was subtractedbased on excitation at 445-490 nm. FIG. 15A shows an in situ carotidbifurcation region image, indicating fluorescence of carotid arterialclot after treatment visible via IVIS imaging after exposure of thecarotid bifurcation zone. FIGS. 15B and 15C are high magnificationimages of fluorescence emanating from the carotid bifurcation in vivo,suggesting accumulation of FNDP in the clot. FIG. 15D is an ex vivophotograph of fluorescence of carotid artery bifurcation denoting onebranch showing fluorescence corresponding to the clot location withinthe carotid bifurcation. FIGS. 15E and 15F are confocal images taken onan Olympus IX83 of FNDP in which fluorescence is detected at anexcitation of 543 nm and an emission of 655-755 nm. Backgroundfluorescence was collected from the same excitation, with emissions of555-625 nm, and was subtracted from the foreground to reduceauto-fluorescence. FIG. 15E shows ex vivo fluorescence of the carotidartery at 4× magnification. FIG. 15F shows FNDP treated carotid arteriesafter they were flushed with RIPA lysis buffer and replicates werecombined together to form a lysate. Lysate was then deposited onto acover-glass and imaged at 20× magnification. Large numbers of FNDP atvarious aggregate sizes around platelets are visible.

FIG. 16 shows fluorescent images taken by IVIS and confocal microscopyof carotid artery clots after intravenous treatment with FNDP. Afterclot formation by ferric chloride, treatment with saline control or FNDPby intravenous infusion via tail vein or femoral vein was performed inthree rats. FNDP were infused (over 10 min) as a suspension in PBS at 1ml solution containing 1 mg/ml FNDP. Carotid arteries were removed fromthe animal for imaging and placed in 70% denatured ethanol forpreservation until imaging. FNDP were conspicuously identified at thesite of clot formation. FNDP were identified in each of the threespecimens obtained following intravenous infusion, yet the threespecimens were treated together as one for lysate inspection. FIGS. 16Aand 16B show imaging of fluorescence as performed on an IVIS scannerdesigned for whole animal imaging using a 580-610 nm excitation and a695-770 nm emission passband with a 2 second exposure. Autofluorescencewas subtracted based on excitation at 445-490 nm. FIG. 8A shows an exvivo fluorescent image of a carotid artery from saline-treated control.Auto-fluorescence could not be entirely eliminated, but was evenlydistributed across control specimen. FIG. 16B shows an ex vivofluorescent image of a carotid artery from an IV FNDP-treated animal,showing fluorescence localized to the branch with a clot. FIGS. 16C-16Fshow confocal images taken on an Olympus IX83. The figures show thatFNDP fluorescence was detected at an excitation of 543 nm and anemission of 655-755 nm. Background fluorescence was collected from thesame excitation, with emissions of 555-625 nm and was subtracted fromthe foreground to reduce auto-fluorescence. FIGS. 16C and 16D show exvivo fluorescence of carotid artery at 4× magnification of salinetreated and FNDP treated animals, respectively. Auto-fluorescence couldnot be entirely eliminated, but was evenly distributed across controlspecimens, while fluorescence was localized to the branch with clot inthe IV treated animal. (See panels 16E and 16F.) Treated carotidarteries were flushed with RIPA lysis buffer and replicates werecombined together to form a lysate. Lysate was then deposited onto acover-glass and imaged at 20× magnification. In order to increasecontrast for visual inspection, images were processed with an un-sharpmask in ImageJ.

FIG. 16E shows that the saline-treated control showed no detectablefluorescence. FIG. 16F shows that FNDP appear as frequent fluorescentspots in the treated samples. FIG. 16G presents a graph showing thenumber of FNDPs present in carotid clot lysates from animals treatedlocally via the external carotid artery or intravenously as comparedwith saline treated controls. FNDPs were counted in replicate imagesafter thresholding in ImageJ.

FIG. 17 depicts images of rat blood plasma clots following treatmentwith F-NDP-Bt and F-NDP-BSA. Rat blood was collected by heart punctureand centrifuged at 100×g for 20 minutes at room temperature to obtainplatelet rich plasma (PRP). A thrombus was generated by adding thrombin(1 U/ml) and incubating for 15 minutes at 37° C. The clot that wasformed was washed 3× by decanting with HBSS containing calcium andmagnesium, and then sliced. Pieces of the clot were incubated with asuspension of F-NDP-Bt and F-NDP-BSA (50 μg/ml) in HBSS containingcalcium and magnesium for 60 minutes at 37° C., and washed 3× with thesame buffer as above, then applied on the glass slide for imaging.Images of plasma clots obtained from fluorescence microscope OlympusIX81analysis, under 100× magnification, are shown in FIG. 17A. Images ofplasma clots obtained using an IVIS 50 imaging system are shown in FIG.17B. Wavelengths used for measurement: excitation Cy5.5 BkG (580-610nm), emission Cy5.5 (695-770 nm). For background subtraction: excitationGFP (445-490 nm), emission Cy5.5 (695-770 nm). Exposure time: 1 minute.Arrows in FIG. 17B point the localization of the clot.

FIG. 17 shows the specificity of interaction of F-NDP-Bt with a clotgenerated from rat blood plasma by thrombin (1 U/ml). Analysis of theclot under a fluorescence microscope (FIG. 17A) revealed that F-NDP-Btaccumulated on the surface of the thrombus to a high extent, althoughthis accumulation was not evenly distributed. Fluorescence microscopyimaging identified areas with high green fluorescence intensity(represented by bright spots in the black and white image), which mayindicate zones of the condensation of activated platelets. Fluorescencelive imaging system (IVIS 50) also exhibited binding of F-NDP-Bt to theplasma clot (FIG. 17B). However, in this system the near infrared (NIR)detection was set up based on the optimization performed as presented inFIG. 9. Control nanoparticles, containing coupled BSA to the surface(F-NDP-BSA), interacted with the clot to a negligible level in bothimaging assays. Detection of F-NDP-Bt by NIR suggested a usefulness offunctionally active F-NDP-Bt for imaging in living organisms because theemission wavelength was localized within an “optical therapeutic window”(600-1300 nm).

Therefore, detection of F-NDP-Bt was performed in a rat model forverification of that hypothesis. The results are shown in FIG. 18. Theskin areas of observation fields were prepared for implantation by hairshaving. The incision was made by scalpel and vessels were insertedunder the skin of dead rats. The dead rats were placed in IVIS 50Imaging System, and measurement of fluorescence under NIR spectrum wasperformed. Wavelengths used for measurement: excitation Cy5.5 BkG(580-610 nm), emission Cy5.5 (695-770 nm). For background subtraction:excitation GFP (445-490 nm), emission Cy5.5 (695-770 nm).

A representative image of the implanted glass capillaries filled withF-NDP-Bt (4 mg/ml) or PBS (control) are shown in FIG. 18A. Exposure timewas 5 seconds. FIG. 10B shows an image of a rat aorta filled withF-NDP-Bt. The rat aorta was dissected from a euthanized female rat,washed with PBS to remove residues of coagulated blood, and filled with300 μl of F-NDP-Bt suspension (2 mg/ml) in PBS. The aorta was securedfrom both ends by knots of surgical sutures. Exposure time was 1 minute.FIG. 18A demonstrates NIR imaging of F-NDP-Bt, experimentally implantedunder rat skin. Suspensions of F-NDP-Bt were infused into glasscapillaries and into dissected rat aorta (FIG. 18B), before subcutaneousimplantation. Clear images showed precise localization of bothartificial (capillary) and natural (aorta) vessels in the rats.

Finally, the specificity of the interaction of F-NDP-Bit with thefibrinogen receptor present on activated platelets in a preformed PRPclot (FIGS. 18C-18D) was investigated. In two separate experiments,clots were incubated with F-NDP-Bit for 15 or 60 minutes, respectively.A clot was generated from rat PRP by thrombin (1 U/mL) and incubatedwith F-NDP-Bit (250 μg/mL) in the presence or absence of Lt (4.67μmol/mL). (FIG. 18C) IVIS imaging was performed using GFP filters(excitation 445-490 nm, emission 515-575 nm). The clot was incubatedwith F-NDP-Bit for 15 minutes (upper panel) or 60 minutes (lower panel).Intensity of fluorescence was evaluated using IVIS Living Image 4.3.1software. Insets above the bars represent respective images of clotsfrom IVIS. (FIG. 18D) Phase-contrast and fluorescence microscope imagesof clots (100×). Areas of accumulation of F-NDP-Bit are framed inyellow. Irrespective of the duration of incubation, binding of F-NDP-Bitwas always 4-5 fold higher than that of the nonspecific controlparticles (F-NDP-BSA). Furthermore, preincubation of the clots withlotrafiban (lt) reduced F-NDP-Bit binding to the level of the controlF-NDP-BSA.

Bitistatin was purified from the venom of Bitis arientans (LatoxanSerpentarium, Valence, France) using two steps of reverse-phase HPLC.F-NDP, chemically surface-functionalized with carboxyl groups (—COOH),were purchased from Adamas Nanotechnologies (Raleigh. N.C., USA). Twostrains of F-NDP were used: green fluorescent F-NDP based on N-V-N colorcenters (F-NDP(NVN)) at 700 nm (2×10⁸ particles/mg) and red fluorescentbased on N-V (F-NDP(NV)) color centers at 100 nm (5×10¹¹ particles/mg),700 nm (2×10⁸ particles/mg), and 10,000 nm (5×10⁵ particles/mg).Isoflurane was purchased from Henry Schein (B34C16A Dublin, Ohio, USA).70% Denatured Ethyl Alcohol and PE-10 tubing were purchased from FisherScientific (Pittsburgh, Pa., USA). Silk Suture was purchased from RobozSUT-15-1, Roboz Surgical Instrument Co. (Gaithersburg, Md., USA).Parafilm and FeCl₃ was purchased from Sigma-Aldrich, (St. Louis, Mo.,USA).

Bitistatin was coupled to the F-NDP of all types using EDC (1-ethyl-3-[3dimethylaminopropyl] carbodiimide hydrochloride) as ahetero-bifunctional cross-linker. Coupling efficiency and preservationof Bitistatin activity on the various functionalized nanodiamondparticles (F-NDP-Bit) were verified using a semi-ELISA methodology.

Example 6

The following example demonstrates the use of functionalized fluorescentnanodiamond particles (e.g., (F-NDP-Bt)) to bind to platelets andpreferentially to activated platelets from humans. The data also showsthat the particles do not substantially interfere with plateletaggregation.

For example, FIGS. 19A-19C show dose response curves for aggregation ofplatelets with and without probe (i.e., F-NDP-Bt), determined as afunction of concentration of proteinase-activated receptor 4 (PAR4 AP),adenosine diphosphate (ADP), and arachidonic acid (AA).

FIGS. 20A-20C show the ability of a F-NDP-Bt probe having an averageparticle diameter of 700 nm or 200 nm to bind to various plateletpopulations at different concentrations. FIGS. 21-222 shows thedifference in binding of probes (700 nm or 200 nm in diameter) instimulated versus unstimulated platelet populations.

Flow cytometry data of stimulated and unstimulated platelets are shownin FIGS. 23A-23D and FIGS. 24A-24D, using the F-NDP-Bt probes.

Example 7

The following example demonstrates an exemplary method for extracting,isolating, and/or quantifying F-NDP-(NV) in blood and/or biologicaltissues.

1. F-NDP-BSA NV 700 nm were prepared using a coupling protocol asdescribed herein, in sterile conditions (see e.g., Example 8). Finalconcentrations of 5 mg/ml were be prepared in PBS for animaladministration.

2. 4 doses of 1 ml (5 mg/ml in PBS) of F-NDP-BSA were injected per onesubject (e.g., an animal). Blood (TBD) may be collected into 3.8% sodiumcitrate (in ratio 1:9) at different time intervals. Control group may beinfused with equal volume of normal saline. Blood from PBS-injectedanimal may be collected in the same way treated samples were processed.At the conclusion of the protocol approximately 10 mL of blood may becollected.

3. Blood containing F-NDP-BSA may be lyophilized using SpeedVac system(SC110A Plus, Thermo Savant) with 4,680×g. Control animal may be usedfor preparation of standard curve. Standard curve may be prepared bymixing known amount of F-NDP-BSA with blood. 8 serial dilution will beprepared starting from the highest, 2 mg/ml particles density.

4. Dry mass of particles containing blood collected in the course of theexperiment of may be dissolved in 12 N HCl at same volume staring volumeof blood (approx. 1 ml). Solubilization may be performed by overnightincubation at 60° C.

5. Supernatant may be removed by centrifugation (17,000×g for 5 min atroom temperature) and pellet re-suspended in the same volume originalvolume (e.g., water 1 ml). Centrifugation will be repeated one time forwashing. At this point, particles may be condensed using smaller amountof water, to improve sensitivity. For example, final volume of water maybe twice lower than volume of blood used for lyophilization, e.g., suchthat sensitivity for detection of F-NDP would be twice increased.

6. Suspension of F-NDP-BSA of investigated samples and standard curvemay be applied on 96-well plate (100 microliters per well) and plate maybe read in Tecan using NIR wavelength (excitation 570 nm, emission 670nm). In parallel, number of particles per ml may be established bydisposable hemocytometer (Incyto Inc., Cheonan-si, Korea), countingunder fluorescence microscope, e.g., using an Olympus IX81 with TRICIwavelength and 400× magnification. Amount of F-NDP-BSA in investigatedsamples may be deducted from the standard curve.

Example 8

The following example demonstrates an exemplary method for sterilizationand/or lyophilization of F-NDPs.

1. F-NDPs were suspended in 70% ethanol and incubate for 15 min at roomtemperature (23+/−2° C.), using gentle agitation on “Speci Mix Test TubeRocker” (ThermoScientific Inc., Waltham, Mass. USA). Density of F-NDPswas approximately 0.5 mg/ml.

2. The suspension of F-NDPs may be centrifuged (e.g., 17,000×g for 5 minat room temperature). Supernatant (ethanol) may be removed by vacuumaspiration and pellet suspended in water or buffer (e.g., PBS or MES)and centrifuged once again under the same speed conditions for washingpurposes.

3. F-NDPs were suspended in water or working buffer (e.g., MES ifcoupling to protein will be performed) in desired density (e.g. 1mg/ml).

4. Lyophilization of F-NDPs may be performed using a SpeedVac system(SC110A Plus, Thermo Savant, and Holbrook, N.Y., USA). For example,polypropylene tubes 5 ml (Sarstedt Inc., Numbrecht, Germany) or 1.5 ml(Fisher Inc. Waltham, Mass. USA) were used for lyophilization. Thissystem generally operates with concentrator set up for working inambient temperature giving low drying rates. For example, application ofhigh drying rates and work in a range of, for example, 43° C.-65° C.temperature may be harmful for some polypeptides and/or polynucleotidesattached to the F-NDP. Concentrator may be used with 8,500 rpm maximalspeed giving 4,680×g maximal force. Vacuum pump (model VLP120, ThermoSavant), may be set up for gas-ballast control “closed” position givingultimate total pressure 1.5×10-3 Torr. Refrigerated vapor trap (modelRVT400, Thermo Savant) may be used with 4 liter chamber capacity giving,approximate operating temperature −50° C.

Lyophilization may be performed from water or buffer suspensiondependent on required purposes (e.g. PBS may be used for in vivoapplication for diagnostic approaches such as thromboembolic events invasculature). Reconstitution of F-NDP may be performed using steriledeionized water, to the desired volume. All procedures may be performedin sterile conditions including all materials such as tubes and tips.

4. Measuring the effect of ethanol sterilization and lyophilization onsize distribution may be performed using Zetasizer Nano ZS (MalvernInstruments Ltd., Westborough, Mass., USA). Working density of F-NDPswas estimated as 10 mg/ml. FIGS. 25A-25B show a high overlap of sameF-NDPs for Particles Size Distribution (Malvern, PSD) assayed before andafter sterilization.

Example 9

The following example demonstrates the ability offluorescent-NanoDiamond particles (F-NDP) covalently conjugated withbitistatin (F-NDP-Bit) to detect vascular blood clots in vivo usingextracorporeal near infrared (NIR) imaging. Specifically, NIRfluorescence properties of F-NDP were compared with color centers (NVvs. NVN) and sizes (100-10,000 nm). Optimal NIR fluorescence and tissuepenetration across biological tissues (rat skin, porcine axillary veinsand skin) was obtained for F-NDP(NV) with a mean diameter of 700 nm.Interavital imaging (IVIS) in vitro revealed that F-NDP(NV)-loaded glasscapillaries could be detected across 6 mm of rat red-muscle barrier and12 mm porcine skin, which to average vertical distance of a humancarotid artery bifurcation from the surface of the adjacent skin (14mm). In vivo, feasibility was demonstrated a rat model ofFeCl₃-generated occlusive blood clots I carotid artery bifurcation.Following systemic infusions of F-NDP(NV)-Bit (3 or 15 mg/Kg) via theexternal carotid artery (ECA) or femoral vein (N=3), presence of theparticles in the thrombi was confirmed both in situ via IVIS, and exvivo, via confocal imaging. F-NDP(NV) presence in the vascular clots wasfurther confirmed by direct counting of fluorescence particles extractedfrom clots following tissue solubilization. The data suggests thatF-NDP(NV)-Bit associate with vascular blood clots, presumably byF-NDP(NV)-Bit binding to activated platelets within the blood clot. Itis posited that F-NDP(NV)-Bit could serve as non-invasive platformtechnology for identification of vascular thrombi using NIR energymonitored by an extra-corporeal device.

Materials

Bitistatin was purified from the venom of Bitis arientans (LatoxanSerpentarium, Valence, France) using two steps of reverse-phase HPLC, asdescribed above. F-NDP, chemically surface-functionalized with carboxylgroups (—COOH), were purchased from Adamas Nanotechnologies (Raleigh.N.C., USA). Two strains of F-NDP were used: green fluorescent F-NDPbased on N-V-N color centers (F-NDP(NVN)) at 700 nm (2×108 particles/mg)and red fluorescent based on N-V (F-NDP(NV)) color centers at 100 nm(5×1011 particles/mg), 700 nm (2×108 particles/mg), and 10,000 nm (5×105particles/mg). Isoflurane was purchased from Henry Schein (B34C16ADublin, Ohio, USA). 70% Denatured Ethyl Alcohol and PE-10 tubing werepurchased from Fisher Scientific (Pittsburgh, Pa., USA). 5-0 Silk Suturewas purchased from Roboz SUT-15-1, Roboz Surgical Instrument Co.(Gaithersburg, Md., USA). Parafilm and FeCl₃ was purchased fromSigma-Aldrich, (St. Louis, Mo., USA).

Coupling of Bitistatin to F-NDP

Bitistatin was coupled to the F-NDP of all types using EDC (1-ethyl-3-[3dimethylaminopropyl] carbodiimide hydrochloride) as ahetero-bifunctional cross-linker. Coupling efficiency and preservationof Bitistatin activity on the various functionalized nanodiamondparticles (F-NDP-Bit) were verified using a semi-ELISA methodology.

Characterization of NIR Emission of F-NDP(NV) and F-NDP(NVN)

NIR fluorescence profiles of F-NDP were characterized using a TecanInfinite 200 PRO (Tecan AG, Mannedorf, CH). 100 μl of 3 mg/ml of 700 nmF-NDP(NV) or F-NDP(NVN) suspended in de-ionized (DI) water were loadedinto 96-well polystyrene. Fluorescence was scanned for all wells withexcitations from 230 nm-850 nm and emissions from 290 nm-850 nm (FIG.1A) at 20 nm intervals. Data was processed in Matlab 2015b (Mathworks,Natick, Mass., USA). Background fluorescence was subtracted from emptywells without F-NDP and the resulting net fluorescence value was Log 10transformed for visualization.

Glass capillaries (40 mm length, 1 mm internal diameter, (Thermo FisherScientific, Waltham, Mass., USA) were filled with equal volumes (30microliters) of suspensions of F-NDP at concentrations from 0.06 and upto 4 mg/ml (1.8-120 g total particle mass) and sealed at each end byplasticine (Hasbro, Pawtucket, R.I., USA). The NIR fluorescenceintensity of the various suspensions in the capillaries were analyzedusing an IVIS 50 Imaging System (PerkinElmer Inc., Akron Ohio) using anexcitation filter set to ‘Cy5.5 BkG’ (580-610 nm) and an emission filterset to ‘Cy5.5’ (695-770 nm) as these filters matched the desired NIRemission profile detected as described above. Imaging was completed with‘binning’ set to 4, and a 10 cm field of view, with exposure timesbetween 2 and 40 seconds. For imaging through biological barriers (ratand porcine skins and rat muscles) auto-fluorescence was imaged with theblue-shifted excitation ‘GFP’ (445-490 nm) and the same emission filter‘Cy5.5’ (695-770 nm) with similar imaging settings as above andsubtracted from the foreground as modified from IVIS 50 protocol tocompensate for the large stokes shift of the F-NDP(NV).12 Thiscorrection was used for a simplified spectral un-mixing: A ratio ofauto-fluorescence between the channel of interest and blue-shiftedexcitation channel is defined in control tissue. The same ratio was thenused to subtract auto-fluorescence from the channel of interest based onthe blue-shifted excitation channel in the test specimen. This operatesunder the assumption that the fluorophore being detected may haveminimal excitation at the blue-shifted wavelength. To assess tissuepenetration of NIR florescence emission from F-NDP, capillaries wereplaced under shaved abdominal rat skin (obtained from euthanized rats),covered with dissected rat quadriceps muscle (2-5.9 mm thick), orcovered with porcine skin (isolated from shoulder of pig obtained from alocal butcher shop)

Generation of Carotid Arterial Blood Clot and F-NDP Infusion in Rat

Technical procedures of the FeCl₃-induced vascular thrombosis model aregenerally known in the art. Specific modifications used in thisparticular work are briefly summarized below. All animal procedures wereperformed according to the guidelines of the US Animal Welfare Act andapproved by the Institutional Animal Care & Use Committee at SUNYDownstate Medical Center. In brief: adult male Sprague-Dawley rats(Charles River, 350 Gm+/−10% body weight), were anesthetized using 4%isoflurane (IF, induction, in chamber) followed by 1-2% IF (maintenance)adjusted throughout the procedure. Rats were held in the supine positionand subjected to surgery using clean instruments and aided bybinoculars. The left carotid artery was dissected and exposed at thebifurcation region. A 5-0 surgical silk suture was wrapped below thecommon carotid (CCA), external carotid (ECA), and internal carotid (ICA)arteries. A PE-10 cannula was then inserted in the ECA for studies whereF-NDP(NV)-Bit were injected locally. A PE-10 cannula was also insertedinto the left femoral vein for studies where F-NDP(NV)-Bit was infusedintravenously (IV). The ICA stem was wrapped in Parafilm soaked in 50%FeCl₃ and kept in place for 10 minutes. Two to three minutes afterplacing the Parafilm onto the ICA, infusion of F-NDP(NV)-Bit suspensionin PBS commenced either via the ECA (N=2, 15 mg/Kg in 1 mL), or via thefemoral vein (N=6) at low dose (N=3, 3 mg/Kg in 1 mL PBS), or at highdose (N=3, 45 mg/Kg in 3 mL PBS). All infusions were completed over 10minutes. Control rats were infused with vehicle at comparable volumesand duration.

Tissue Fixation Post F-NDP(NV)-Bit Infusion

Following the completion of particles infusion, anesthesia was augmentedto produce deep hypnosis using 5% IF. The lower aorta was quicklyisolated and cut to allow blood drainage. Tissue was fixed and residualblood removed by perfusion with 10 mL of 70% denatured ethanol.Dissection of both bilateral carotid artery bifurcation regions wascompleted after whole body imaging (IVIS). Vessels were suspended 70%denatured ethanol for further ex vivo NIR fluorescence evaluation.

In Situ and Ex Vivo Imaging of F-NDP(NV) Fluorescence by IVIS

Briefly, NIR fluorescence was detected using a 580-610 nm excitation anda 695-770 nm emission pass-band with 2 second exposure, ‘binning’ set to4, and a 7 cm field of view as described above. Auto-fluorescence wassubtracted based on excitation at 445-490 nm under otherwise similarimaging conditions. Carotid arteries were exposed before imaging toenable clear visualization for in situ images. Following in situimaging, carotid arterial bifurcations were removed from animals andplaced on a glass plate for imaging ex vivo using identical imagingparameters to those used for in situ imaging. For each artery, the meanfluorescence intensity of the images compensated for auto-fluorescencewas calculated using ImageJ (NIH, Bethesda, Md., USA).

Ex Vivo Imaging of F-NDP(NV) by Fluorescence Microscopy

Gross images of the entire carotid bifurcation region extracted fromclot bearing or contralateral vessels were evaluated on a FluoviewFV1000 (Olympus, Tokyo, Japan) laser scanning confocal microscope (LSCM)using a 4× objective. NIR fluorescence emitted from F-NDP(NV) wasdetected at an excitation of 543 nm and an emission of 655-755 nm.Confocal stacks were combined using a maximum intensity projection sothat the entire vessel is brought into focus. The mean fluorescenceintensity of F-NDP in each artery was calculated after subtraction ofthe local background using ImageJ.

Isolation of F-NDP(NV) from Vascular Clot

F-NDP were isolated from extracted carotid arteries by homogenization inRIPA lysis buffer (Teknova Inc. Hollister, Calif., USA) at 100 mg/ml.Aliquots (10 microliters) of the lysate suspension were applied on themicroscope slides and analyzed under LSCM as above under 20× objective.

In animals treated with F-NDP(NV) via the femoral artery at high dose,F-NDP(NV) were isolated from extracted carotid arteries by solubilizingthe clot bearing vessel segment in 12 N hydrochloric acid (HCl) (ThermoFisher Scientific, Waltham, Mass., USA) overnight at 60° C. at 100mg/ml. The solution was centrifuged (14,000×g at room temperature for 10minutes) and the pellet was washed 1× with distilled water. The pelletcontaining the insoluble F-NDP(NV)-Bit was re-suspended in DI-waterwhile keeping the initial mass/volume ratio. An aliquot of thesuspension was applied to a hemocytometer (Incyto Inc., Cheonan-si,Korea), which was standardized for particles counting in an invertedfluorescence microscope (Olympus IX81) with 40× objective. Images ofF-NDP(NV)-Bit were taken from each observation field for counting usingTRITC filter cube. Numbers of particles were calculated for the entiresolubilized tissue.

Statistical Analysis

Unless stated otherwise, each experiment in this example was performedindependently three times in triplicate. No outlying data was excluded.Data are represented as mean±SD. Statistical analyses were done by theStudent's t test using (SigmaPlot® 12 SPSS, Systat Software Inc., SanJose Calif., USA). P<0.05 was considered significant.

Results

Comparison of NIR fluorescence intensity of F-NDP(NVN) and F-NDP(NV)fluorescence profile measurements revealed NIR fluorescence in both N-Vand N-V-N particles. However, fluorescence in the NIR region was 20times greater in the N-V particles (FIG. 26A). This experiment alsorevealed the peak excitation of F-NDP(NV) at 570 nm and peak emission at670 nm. Using this peak excitation and emission profile, NIR emission ofthe F-NDPs was compared in the IVIS in a dose response manner (FIG. 26B,FIG. 26C). Capillary studies revealed that under the same excitationconditions, NIR emission of the F-NDP(NV) was more effective than thatof F-NDP(NVN) by approximately an order of magnitude (FIG. 26B).

Fluorescent characteristics of F-NDP may vary with size of particle. TheNIR emission of identical mass per mL of F-NDP(NV) was compared forparticles of three different sizes (FIGS. 27A-27D). The lowestfluorescence intensity was observed for smallest (100 nm) particles,while the highest fluorescence emission was observed for 700 nm F-NDP.Fluorescence scaled linearly with exposure time revealing no bleachingat the higher exposure times tested. While fluorescence from the 700 nmF-NDP(NV) particles saturated the detector in less than 20 seconds,longer exposure times were required to clearly display fluorescence fromthe F-NDP(NVN) particles. It is noteworthy that for the same acquisitiontime the NIR emission of 700 nm and 10,000 nm was not augmented. Infact, at 0.5 mg/ml and 1 mg/ml, the emission of the 10,000 nm particles,was significantly lower than that of the 700 nm particles (p<0.01, FIG.27A). We then tested the ability of NIR fluorescence emitted fromequivalent sized (700 nm) F-NDP particles of the N-V and N-V-N strainsof F-NDP to penetrate biological barriers as imaged in the IVIS (FIGS.28A-28H). Capillaries filled with 4 mg/ml F-NDP(NV) particles could beimaged through rat skin (FIG. 28A) or quadriceps muscle (FIG. 28B) aswell as porcine axillary vein (FIG. 28C) and 2.5 mm of defatted porcineskin (FIG. 28D), while capillaries filled with F-NDP(NVN) particlescould not be visualized with similar imaging parameters in any of thesecircumstances. NIR fluorescence was monitored through 2.5 mm of defattedporcine skin at concentrations from 1-4 mg/ml (30-120 micrograms total)(FIG. 28E) as the signal did not penetrate full-thickness tissue.Porcine axillary veins loaded with 1 mL of 2 mg/mL of F-NDP(NV)particles could be visualized through 8 mm of porcine skin (FIG. 28F).As a final test of penetration capacity, an angled piece of fullthickness porcine skin varying from 9 to 14 mm in thickness was laid ontop of capillaries containing 20 mg/mL (600 g total) of F-NDP(NV). Adetectable signal from the F-NDP(NV) was recorded through the porcineskin up 12 mm in thickness. In contrast, no light emission was detectedat equal conditions of F-NDP(NVN) (FIG. 28G). Noteworthy is dataobtained by ultrasound imaging of human carotid arteries, where thecarotid artery bifurcation distance from the skin surface was assessedat 14 mm below the skin surface. These findings suggest a translationalprospect of F-NDP(NV) to detect blood clot in this area if comparableparticle mass can be safely deposited on a blood clot in this region(FIG. 28H).

Detection of Blood Clot in Rat Model Using F-NDP(NV)-Bit

The results depicted in FIGS. 26A-28H suggest that F-NDP(NV) (700 nm)are the brightest of the particles tested and may be a useful F-NDPstrain for imaging in vivo. Therefore, all subsequent in vivo studieswere carried out with F-NDP(NV) coupled with Bit for detection ofthrombi generated in the carotid artery bifurcation of rats. After clotformation and treatment with F-NDP(NV)-Bit via the ECA, carotid arterieswere imaged in situ and removed from the animal for imaging andanalysis. Injection of F-NDP via the ECA optimizes the exposure of theparticles to the lesion site thus avoiding potentially confoundingvariables of distribution, uptake, and elimination. Imaging offluorescence in the IVIS scanner demonstrated strong fluorescence insitu (FIG. 29A, FIG. 29B) in the vessel branches corresponding to thelocation of the clot in the exposed artery. After removing the carotidarterial bifurcations from treated and untreated animals, a strongfluorescent signal is detected (FIG. 29C, FIG. 29D vs FIG. 29E, FIG.29F) in the FeCl₃-treated arteries. This was further validated byconfocal imaging confirming co-location of clot and deposited particles(FIG. 29G, FIG. 29H vs FIG. 29I, FIG. 29J). As a final validationmethod, particles were imaged in pooled lysates (2 lesions), showinglarge numbers of fluorescence particles in F-NDP(NV)-treated animals ascompared to their absence in animals treated with vehicle (FIG. 29K).Following in the initial proof of feasibility direct administration ofthe F-NDP(NV)-Bit via ECA, a low (3 mg/Kg) and high (15 mg/kg) dose ofF-NDP(NV)-Bit were infused systemically into animals via the femoralvein. The results from animals treated with the low dose wereinconclusive, as the signal was not consistently above the fluorescencelevel detected from carotid bifurcation isolated from control animalstreated with vehicle only. Despite this, pooled lysates (3 lesions)showed that F-NDP particles had been logged in the clots, while noparticle associated fluorescence was detectable from vehicle treatedanimals (FIG. 29L).

After IV injection of the high dose of F-NDP(NV)-Bit, clot-associatedfluorescence was demonstrated in situ in all treated animals (FIGS.30A-30C). Arteries with FeCl₃-generated lesions and the contralateralcontrol were then dissected and imaged independently by IVIS andconfocal microscopy. A strong fluorescence signal in the treated arterywas observed by both IVIS (FIGS. 30D-30F vs FIGS. 30G-30I) and confocalimaging (FIGS. 30J-30L vs FIGS. 30M-30O). The difference in brightnessto that of the control artery was statistically significant (FIG. 30R,FIG. 30S) with p<0.05. Lysates collected by dissolving the carotidarteries also demonstrated a large number of fluorescent particlesresiding in the lesioned artery and a low, but consistent, number ofparticles in the contralateral control arteries (FIG. 30P). Thisdifference was highly statistically significant (FIG. 30Q) with p<0.01.

The two main objectives of the example described above were 1)characterization of the two strains of the F-NDP (F-NDP(NV) andF-NDP(NVN)) in terms of their NIR emission parameters and 2)identification of the F-NDP strain, which emits sufficient energy thatmost likely penetrates biological tissues over a distance required forthe translational application in human NIR vascular pathology imaging.Given the prospect of longer (minutes) scale of imaging procedures inhumans an important consideration in selection F-NDP includes thestability/durability of the NIR emission. Nanoparticles in general tendto have reduced toxicity with increased diameter. Specifically,nanodiamonds display reduced toxicity (as compared to e.g. nanotubes)with increasing diameter (tested up to 100 nm), suggesting that the 700nm F-NDP(NV) used in this study may be safe. Preliminary studies suggestno mortality or morbidity in rats injected via venous port, with 700 nmF-NDP(NV) at 45 mg/kg while evaluated for weight and neurobehavioraltests for up to 5 days.

The first objective was to systematically investigate the emissionattributes each of F-NDP across four independent variables: a) thefluorescence spectrum and brightness resulting from the atomicmanipulations (N-V, N-V-N); b) particles total mass relationships toemission intensity; c) the relationship of particle diameter to emissionintensity and d) speed and extent of NIR acquisition kinetics. Thesevariables have been exercised ‘head to head’ between N-V and N-V-Nstrains using IVIS technology, which were considered all well suited forsuch comparison in due to its sensitivity and its non-invasive NIR lightdetection capability. The data presented in FIGS. 26A-27D point to theprospect of the F-NDP(NV) strain as useful for in vivo NIR fluorescentpenetration through biological tissues. For example, in this example the700 nm F-NDP(NV) exhibited 10-60 higher NIR emission intensity than theF-NDP(NVN). Of note, NIR emission from F-NDP(NV) was not directlycorrelated with particle size, and was maximized for 700 nm F-NDP(NV).At constant particle mass loading, the 700 nm particles were ˜4 timesbrighter in IVIS images, and consistently and across all acquisitionperiods tested; NIR fluorescence emission from 10,000 nm particles wasweaker than from e 700 nm particles (FIG. 2). While 10,000 nm F-NDP(NV)may be not likely to be useable for IV injection, the 10,000 nmparticles demonstrated that under equal mass conditions particlebrightness was not correlated with diameter, but may be maximized forparticles of a specific diameter range.

Commensurate with the physical properties delineated for the F-NDP(NV)(FIGS. 26A-27D) the data in FIGS. 28A-28H further support the potentialof the F-NDP(NV) to support in vivo vascular clot imaging. FIG. 3presents several conditions where tissues penetration of NIRfluorescence emitted from F-NDP(NV) was tested. The maximum distancedetectable by NIR through porcine skin, used as a human skin analog,(FIG. 28G) was 12 mm. FIG. 28H presents an ultrasound recording ofnormal human carotid artery bifurcation annotated for the ICA, ECA andCCA. The linear dashed bar in FIG. 28H indicates 11.89 mm depth of thehuman carotid artery bifurcation from the vertical distance from theneck surface. Considering the dense epidermis of porcine shoulder skinas compared to human neck and the distance of the IVIS camera from thetarget to be monitored versus the use of a similar hand-held deviceplaced directly on the skin, it is posited that NIR fluorescent imagingof a clot bin the carotid artery bifurcation is likely to be within anachievable diagnostic opportunity. Furthermore, NIR fluorescencerecorded in FIG. 28H represents a source generated from ˜670 ug of 700nm F-NDP(NV) particles. The same amount of particles tagged onto humanclot in this region could enable clot detection by NIR fluorescenceimaging. Results in FIG. 30Q indicates a minimum of 0.7% of injecteddose was captured in the lesion of interest (2×10⁸ particles/mg indose), which would imply a required dose of approximately 100 mg (1.4mg/kg) to reach a similar emission profile in human. In preliminarystudies in rat, doses as high as 45 mg/kg were well tolerated for 5 dayswithout any adverse events recorded (data not shown).

In vivo studies were performed with an F-NDP(NV)-Bit covalently coupledwith bitistatin. The procedure used EDC-mediated covalentheterobifunctional coupling, yielding a stable amide bond, which isresilient in biological systems. These F-NDP(NV)-Bit, were administeredsystemically to anesthetized rats subjected to an established carotidartery clot procedure at the site of the bifurcation. In the pilottranslational study F-NDP(NV)-Bit were first administered via the ECA(‘high dose’, 15 mg/Kg) or via the femoral artery (‘low dose’, 3 mg/Kg).This experimental design was selected since pharmacokinetics andparticles distribution dynamics are as yet unknown. Therefore, to avoidpossible loss of significant amount of particles via ‘first path’elimination or tissue distribution (potential ‘false negative’ outcome),injection via the ECA ascertains maximum exposure of the particles tothe clot in the targeted region. To assess and confirm specificco-localization of F-NDP(NV)-Bit in the blood three independent methodswere deployed: a) IVIS total body imaging (FIG. 29A, FIG. 29B); b) LSCMof extracted vessels carrying clots (FIG. 29C, FIG. 29D) and c) directcount of particles extracted from clot-bearing vessels aftersolubilization of all organic material (FIG. 29L). Association ofF-NDP(NV) by infusion of a high dose of F-NDP(NV)-Bit via the ECA iseasily detected by all three methods. However, infusion of a low dose (3mg/Kg) of F-NDP(NV)-Bit via the femoral vein failed to detect emissionby either IVIS or by LSCM (FIG. 4E-F). A low amount of particles wascounted in the clot extract (FIG. 29M), yet it is not clear whether thisvery small number is specifically clot associated, loaded into thevessel wall via “vasa-vasorum” or both. It is clear however that the lowdose of F-NDP(NV)-Bit did not produce a credible emission signal thatcould be detected by IVIS or LSCM, even if particles were in fact targetto the clot. Next, F-NDP(NV)-Bit was intravenously administered at ahigher dose (15 mg/Kg, N=3). As illustrated in FIGS. 30A-30C, all threeanimal tested displayed a strong fluorescence signal emanating from thecarotid bifurcation zone (IVIS), as also clearly visible in thefluorescence of isolated vessels (FIGS. 30D-30I) and under inspection byLSCM (FIGS. 30J-30O). Particles were also present in large numbers insolubilized clot-bearing vessels (FIG. 30P). FIGS. 30Q-30S show thequantitative analysis of the robust deposition of particles inclot-bearing vessels vs. the control contralateral.

In summary in this example evidence was demonstrated that the F-NDP(NV)deployed in this study can associate with clot in vivo, such as athrombus formed in a rat carotid artery bifurcation model. Theproof-of-concept here is based on three independent measures or the NIRfluorescence detected at the in situ clot formation, including directcounting of fluorescence particles isolated from the extracted clot. Thedata demonstrates the possibility to detect by NIR fluorescence imagingemitted from F-NDP(NV) over a distance corresponding to that present invascular pathology (e.g. clot in the carotid artery bifurcation). Ifsuccessfully translated to clinical practice, this minimally invasiveprocedure, conducted in ambulatory settings, could enhance preventativemeasures, such as earlier initiation of anti-thromboembolic medications.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,and/or methods, if such features, systems, articles, materials, and/ormethods are not mutually inconsistent, is included within the scope ofthe present invention.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively, asset forth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

1. A diagnostic agent, said agent comprising a fluorescent nanodiamondparticle chemically bonded to a polypeptide, ligand, and/orpolynucleotide, wherein the fluorescent nanodiamond particle has anaverage cross-sectional dimension of greater than or equal to 250nanometers and less than or equal to 1000 nanometers. 2-4. (canceled) 5.The diagnostic agent of claim 1, wherein the fluorescent nanodiamondparticle and the polypeptide, ligand, and/or polynucleotide arecovalently bonded. 6-31. (canceled)
 32. A diagnostic agent as in claim1, wherein the polypeptide is a disintegrin.
 33. (canceled)
 34. Adiagnostic agent for detection of thrombotic events in a human ornon-human animal, said agent comprising a nanodiamond particlechemically bonded to a disintegrin. 35-36. (canceled)
 37. The diagnosticagent of claim 34, wherein the nanodiamond particle and the disintegrinare covalently bonded. 38-46. (canceled)
 47. A diagnostic agent as inclaim 34, wherein the nanodiamond particle has an averagecross-sectional dimension of greater than or equal to 250 nanometers andless than or equal to 1000 nanometers.
 48. A diagnostic agent as inclaim 1, wherein the polypeptide is a protein, an antibody, and/or anantigen.
 49. A diagnostic agent as in claim 32, wherein the disintegrinis selected from the group consisting of albolabrin, applagin,barbourin, batroxostatin, bitistatin, obtustatin, schistatin,echistatin, elegantin, eristicophin, flavoridin, halysin, kistrin,mojastin, rubistatin, tergeminin, salmosin and triflavin.
 50. Adiagnostic agent as in claim 1, wherein the polypeptide orpolynucleotide is covalently bound to the plurality of fluorescentnanodiamond particles.
 51. A diagnostic agent as in claim 1, wherein theplurality of fluorescent nanodiamond particles comprise anatomistic-type defect.
 52. A diagnostic agent as in claim 51, whereinthe atomistic-type defect is a nitrogen-vacancy defect, anitrogen-vacancy-nitrogen defect, or an Si-vacancy defect.
 53. Adiagnostic agent as in claim 1, wherein the plurality of fluorescentnanodiamond particles are intrinsically fluorescent.
 54. A diagnosticagent as in claim 34, wherein the nanodiamond particle is intrinsicallyfluorescent.
 55. A diagnostic agent as in claim 34, wherein thedisintegrin is selected from the group consisting of albolabrin,applagin, barbourin, batroxostatin, bitistatin, obtustatin, schistatin,echistatin, elegantin, eristicophin, flavoridin, halysin, kistrin,mojastin, rubistatin, tergeminin, salmosin and triflavin.
 56. Adiagnostic agent as in claim 34, wherein the disintegrin is covalentlybound to the plurality of nanodiamond particles.
 57. A diagnostic agentas in claim 34, wherein the plurality of nanodiamond particles comprisean atomistic-type defect.
 58. A diagnostic agent as in claim 57, whereinthe atomistic-type defect is a nitrogen-vacancy defect, anitrogen-vacancy-nitrogen defect, or an Si-vacancy defect.